ES2978086T3 - Aspartoacylase gene therapy in the treatment of Canavan disease - Google Patents

Abstract

In some aspects, the disclosure relates to compositions and methods useful for the diagnosis and treatment of neurodegenerative diseases, such as leukodystrophies (e.g., Canavan disease). In some embodiments, the methods comprise administering to a subject an N-acetylaspartate (NAA)-reducing agent or an N-acetylaspartate (NAA)-reducing agent based on the subject's metabolic profile. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Aspartoacylase gene therapy in the treatment of Canavan disease

BACKGROUND

Central nervous system (CNS) disorders represent a huge economic burden on society, with increasing incidence and prevalence in populations worldwide. Neurodegenerative disorders are a subgroup of CNS disorders caused by a variety of genetic and nongenetic factors with variable disease onset, e.g. Canavan disease, Alzheimer's disease, or multiple sclerosis (MS). A subgroup of neurodegenerative disorders are leukodystrophies, which are diseases affecting the white matter of the CNS. The white matter of the CNS is composed of oligodendrocytes that form the myelin that surrounds neuronal axons. One function of oligodendrocytes is to facilitate the propagation of axonal potential.

WO 2015/127128 describes recombinant adeno-associated viruses (rAAVs) having various tissue targeting capabilities, including AAV capsid proteins and the use of rAAVs for gene transfer methods. US 2014/335054 relates to rAAVs useful for targeting transgenes to the CNS, for example, to treat CNS-related disorders. US 2014/142152 describes methods and compositions for treating cancer, including cancers in which aspartocylase (ASPA) and/or N-acetylaspartate (NAA) expression or activity is decreased. US 2013/0023488 provides compounds for reducing lipid accumulation in a cell that are useful for treating lipid/glycogen disorders. Patent document EP 2261242 relates to the identification of the NAA-forming enzyme aspartate-N-acetyltransferase and its use in diagnosis and drug screening, for example in neurological disorders.

SUMMARY

The invention is explained in the appended claims.

The disclosure relates, in some aspects, to compositions and methods useful for diagnosing and treating neurodegenerative diseases. In some aspects, the disclosure relates to the discovery that impaired N-acetylaspartate (NAA) metabolism or aspartoacylase (ASPA) deficiency shifts energy metabolism in the CNS away from glycolysis and toward beta-oxidation (e.g., fatty acid metabolism) in subjects suffering from white matter diseases (e.g., Canavan disease), or other neurodegenerative disorders, such as Alzheimer's disease and traumatic brain injury. Without wishing to be bound by any particular theory, the methods and compositions described herein identify and/or correct metabolic imbalances in the CNS of a subject suffering from a neurodegenerative disease.

Aspects of the disclosure relate to methods of treating leukodystrophy in a subject in need thereof. The methods may comprise administering to the subject an N-acetylaspartate (NAA) reducing agent. The leukodystrophy may have been determined to be associated with a metabolic imbalance comprising a shift from glycolysis to beta-oxidation in the subject. The methods may further comprise detecting the metabolic imbalance by assessing levels of one or more glycolysis and/or beta-oxidation factors (e.g., by assessing levels of a reporter molecule or set of molecules of a metabolic pathway, e.g., one or more of those listed in FIG. 57). The levels of one or more glycolysis and/or beta-oxidation factors may be determined using CNS fluid obtained from the subject.

Treatment for leukodystrophy may involve obtaining CNS fluid from a subject; detecting increased beta-oxidation in the CNS fluid; and, based on the detected increase in beta-oxidation, administering to the subject an N-acetylaspartate (NAA)-reducing agent. The NAA-reducing agent may be ASPA.

Treatment for leukodystrophy may include measuring a metabolic profile of a biological sample obtained from a subject; identifying a metabolic imbalance associated with leukodystrophy based on the metabolic profile; and administering to the subject an N-acetylaspartate (NAA)-reducing agent. The metabolic imbalance may involve a shift from glycolysis to beta-oxidation.

A leukodystrophy may be associated with a condition selected from the group consisting of Canavan disease, adrenomyeloneuropathy, Alexander disease, cerebrotendinous xanthomatosis, Krabbe disease, metachromic leukodystrophy, adrenoleukodystrophy, Pelizaeur disease, and Refum disease. In some embodiments, a leukodystrophy is associated with Canavan disease.

Metabolic profiling may involve analysis of the biological sample using liquid chromatography (LC), mass spectrometry (MS), or liquid chromatography/mass spectrometry (LC/MS). Metabolic profiling may involve analysis of the biological sample using ultra-high performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS).

The biological sample may include CNS tissue or cerebrospinal fluid (CSF). CNS tissue may include brain tissue.

A metabolic profile may comprise a level of a first biomarker selected from the group consisting of glucose, glucose-6-phosphate, 3-phosphoglycerate, pyruvate, lactate, and phosphoenolpyruvate. A metabolic profile may comprise a level of a second biomarker selected from the group consisting of carnitine, malonylcarnitine, myristoylcarnitine, palmitoylcarnitine, malonylcarnitine, and beta-hydroxybutyrate. A metabolic profile may further comprise a level of one or more additional biomarkers indicating a reduction in the subject's glycolysis. The metabolic profile may further comprise a level of one or more additional biomarkers indicating an increase in the subject's beta-oxidation.

An NAA-reducing agent may be selected from the group consisting of a small molecule, a protein, and a nucleic acid. In some embodiments, an NAA-reducing agent is delivered using a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises: a capsid protein; and, a nucleic acid comprising a promoter operably linked to a transgene, e.g., a transgene encoding aspartoacylase (ASPA). In some embodiments, the promoter is an astrocyte-specific promoter. In some embodiments, the astrocyte-specific promoter is the glial fibrillary acidic protein (GFAP) promoter.

In some embodiments, a capsid protein has an AAV9 serotype.

In some embodiments, the rAAV is administered by injection. In some embodiments, the injection is selected from the group consisting of intravenous injection, intravascular injection, and intraventricular injection. In some embodiments, the administration results in expression of the gene in peripheral tissue. In some embodiments, the administration results in expression of the gene in CNS tissue. In some embodiments, the administration results in astrocyte-restricted expression of the gene.

The methods provided herein may further comprise administering to the subject a small molecule metabolic modulator. The methods provided herein may further comprise prescribing to the subject a dietary intervention, wherein the dietary intervention promotes glycolysis and/or reduces beta-oxidation in the subject. The methods provided herein may further comprise administering to the subject an immunosuppressive agent. The immunosuppressive agent may comprise prednisone or a corticosteroid. The immunosuppressive agent may be administered to the subject prior to administration of the rAAV.

Aspects of the disclosure relate to methods for treating a neurodegenerative disease. The methods may comprise: measuring a metabolic profile of a biological sample obtained from a subject; identifying a metabolic imbalance associated with the neurodegenerative disease based on the metabolic profile; and, administering to the subject an N-acetylaspartate (NAA) increasing agent. The metabolic imbalance may comprise a decreased level of N-acetylaspartate (NAA). The methods may involve assessing the levels of a reporter molecule or a set of molecules of a metabolic pathway to establish a metabolic profile. Neurodegenerative diseases may be selected from the group consisting of Alzheimer's disease, traumatic brain injury (TBI), bipolar disorder, catalepsy, epilepsy (e.g., seizures), migraine, Huntington's disease, attention deficit disorder (ADD), attention deficit/hyperactivity disorder (e.g., ADHD), autism spectrum disorder (e.g., Asperger's disease, autism, etc.), Parkinson's disease, Tourette syndrome, clinical depression, multiple sclerosis, and autoimmune disease (e.g., CNS demyelinating disease, myasthenia gravis, etc.). Metabolic profiling may comprise analysis of the biological sample using liquid chromatography (LC), mass spectrometry (MS), or liquid chromatography/mass spectrometry (LC/MS). Metabolic profiling may comprise analysis of the biological sample using ultra-high performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). The biological sample may include CNS tissue or cerebrospinal fluid (CSF). CNS tissue may include brain tissue.

The metabolic imbalance may not be caused by an ASPA deficiency. The metabolic profile may comprise a level of one or more biomarkers that indicate a change in the subject's glycolysis. The metabolic profile may comprise a level of one or more biomarkers that indicate a change in the subject's beta-oxidation.

In some of the methods described herein, an N-acetylaspartate (NAA)-enhancing agent can be selected from the group consisting of a small molecule, a protein, and a nucleic acid. The N-acetylaspartate (NAA)-enhancing agent can be administered using a recombinant adeno-associated virus (rAAV). The rAAV can comprise: (a) a capsid protein; and, (b) a nucleic acid comprising a promoter operably linked to a transgene, for example, a transgene encoding N-acetylaspartate synthetase (NAT8L). The capsid protein can have a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, and AAV.rh10. The rAAV can be administered by injection. The injection may be selected from the group consisting of intravenous injection, intravascular injection, and intraventricular injection. The administration may result in ubiquitous expression of the transgene. The administration may result in expression of the gene in peripheral tissues. The administration may result in expression of the gene in CNS tissue. The methods may further comprise administering a small molecule metabolic modulator to the subject.

Other aspects of the disclosure relate to methods for increasing ATP production in a subject. The methods may involve administering to a subject a recombinant adeno-associated virus (rAAV) comprising a transgene encoding the ASPA enzyme, or the NAT8L enzyme. In some aspects, the subject does not have an ASPA deficiency or a neurodegenerative disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data illustrating that rAAV-ASPA gene therapy restores the thalamocortical tract, as measured by diffusion tensor magnetic resonance imaging (DTI) for cerebral water flow.

FIG. 2 shows data illustrating that Canavan disease (CD) causes increased oxygen consumption for functional neuroconnectivity, as measured by resting-state functional MRI (RS-fMRI).

FIG. 3 shows data illustrating that Canavan disease (CD) causes increased oxygen consumption for cortical-subcortical connectivity.

FIG. 4A-4B show the statistical analysis of the whole-brain metabolome in wild-type (WT) and ASPA gene knockout (KO) mice. Treated mice were administered rAAV-ASPA via intravenous injection at P1. Neurometabolome data were analyzed at P25; N=8. FIG. 4A shows a Principal Component Analysis (PCA) of WT (untreated/treated) and KO (untreated/treated) mice. FIG. 4B shows the Hierarchical Cluster Analysis of WT (untreated/treated) and KO (untreated/treated) mice. Both FIG. 4A and 4B show the clustering of WT and KO (treated) mice, indicating that rAAV-ASPA treatment rescues the metabolic phenotype in CD mice.

FIG. 5 shows representative data relating to glucose metabolism biomarker levels in WT (untreated and treated) and KO (untreated and treated) mice. Treated mice were administered rAAV-ASPA.

FIG. 6 shows representative data relating to beta-oxidation biomarker levels in WT (untreated and treated) and KO (untreated and treated) mice. Treated mice were administered rAAV-ASPA.

FIG. 7 shows representative data illustrating the downregulation of O<2> radical scavengers (e.g., anserine) and their precursor molecules (e.g., cysteine) in CD (KO) mice. Treatment with rAAV-ASPA restores anserine and cysteine levels in KO mice.

FIG. 8 shows data illustrating that CNS-restricted (intraventricular, ICV) and systemic (intravenous, IV) administration of rAAV-ASPA results in comparable therapeutic outcomes in CD mice treated at P1. Mice were evaluated at P26.

FIG. 9 shows data illustrating that rAAV-ASPA administration restores mobility to Nur7 mice (a model of mild CD). rAAV-ASPA was administered to mice at various time points (e.g., 1 month of age, 2.5 months of age, 6.5 months of age). Psychomotor function was assessed using rotating cylinder and balance beam tests.

FIG. 10 shows data illustrating that cognitive function (e.g., working/spatial memory) is restored in Nur7 mice intravenously administered rAAV-ASPA at P1 and at 3 months.

FIG. 11 shows data illustrating rapid and efficient clearance of CNS spongy degeneration in Nur7 mice receiving intravenous administration of rAAV-ASPA at P1. Neuropathology was assessed at P25.

FIG. 12 shows data illustrating that administration of rAAV-ASPA rapidly reduces NAA in the Nur7 mouse brain. Nur7 mice were treated with rAAV-ASPA at 6 weeks of age and monitored at 7 and 10 weeks of age by neuroimaging.

FIG. 13 shows data illustrating that rAAV-ASPA treatment restores the myelinolipid profile in CD mice. Compared to WT mice, KO mice exhibit significantly reduced levels of sphingolipids and other myelin components. rAAV-ASPA-treated CD mice exhibit a significant increase in myelin components such as sphinganine.

FIG. 14 shows the weight loss/gain patterns in wild-type (WT) and CD model (Nur7) mice. Nur7 mice treated by i.v. administration of 3rd generation rAAV-hASPA (FKzhAspA-Opt) at P1 show similar growth as WT mice. Male and female mice show the same pattern of weight loss/gain.

FIG. 15 shows the improvement in overall health of Nur7 mice treated with rAAV-hASPA. Nur7 mice were treated with rAAV-hASPA at p1, p42, p84, or p168 and weighed. Treated mice were compared to a wild-type (WT) mouse control. All treatment groups show normalization/improvement in weight.

FIG. 16 shows data regarding the expanded therapeutic window for the treatment of CD using rAAV-hASPA. Nur7 mice were treated with rAAV-hASPA at p1, p42, p84, and p168, and motor function was assessed by roller-roller. Mice treated at 6 weeks of age fully recovered within 4 weeks postinjection.

FIG. 17 shows data regarding the expanded therapeutic window for the treatment of CD using rAAV-hASPA. Nur7 mice were treated with rAAV-hASPA at p1, p42, and p84, and motor function was assessed using the balance beam.

FIG. 18 shows data related to the assessment of cognitive function in rAAV-hASPA-treated Nur7 mice. At p70, rAAV-hASPA-treated Nur7 mice outperformed wild-type (WT) mice with respect to total distance traveled and total distance traveled in the periphery.

FIG. 19 shows normalization of T2 signal and NAA levels in the brains of Nur7 mice treated with rAAV-hASPA, as shown by magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and measurement of NAA aciduria.

FIG. 20 shows normalization of brain morphology at p25 in Nur7 mice treated with rAAV-hASPA at p1.

FIG. 21 shows complete normalization of T2 and ERM signal intensities in one-year-old Nur7 mice that were treated with rAAV-hASPA at p42.

FIG. 22 shows improved overall health, as measured by weight gain, in CD KO mice treated with astrocyte-restricted rAAV-hASPA. Astrocyte-specific ASPA expression was produced using a glial fibrillary acidic protein (GFAP) promoter to drive hASPA expression. The lifespan of CD KO mice treated with astrocyte-restricted rAAV-hASPA exceeded the 28-day lifespan of untreated KO mice and was not significantly different from the lifespan of wild-type (WT) mice.

FIG. 23 shows that astrocyte-restricted hASPA expression results in normalization of motor function in CD KO mice. Astrocyte-restricted rAAV-hASPA was administered to CD KO mice and motor function was measured at p27 and p90. The data show that astrocyte-restricted hASPA expression resulted in restoration of motor function in treated CD KO mice compared to wild-type (WT) mice. At p90, treated CD KO mice outperformed WT mice in a rotating cylinder test.

FIG. 24 shows that astrocyte-restricted hASPA expression results in normalization of motor function in CD KO mice. CD KO mice were administered astrocyte-restricted rAAV-hASPA and motor function was measured by balance beam test at p27 and p90. The data show that astrocyte-restricted hASPA expression resulted in restoration of motor function in treated CD KO mice compared to wild-type (WT) mice.

FIG. 25 shows that astrocyte-restricted hASPA expression normalizes NAA levels, as measured by ERM.

FIGS. 26A-26C show that the optimized gene expression cassette rescues normal ASPA and NAA protein levels in CD KO mice. FIG. 26A shows that three expression cassettes carrying either the full-length (FKz) or half (HKz) Kozak sequence and either wild-type (WT) human aspartoacylase (hASPA) cDNA or a codon-optimized hASPA (Opt) were cloned; the 3rd generation construct comprises full-length hASPA FKz and Opt. FIG. 26B shows that mice were treated at p1 via the facial vein with 4x10″ genomic copies (CG) of rAAV9 carrying an expression cassette from either the 1st, 2nd, or 3rd generation. WB of brains 42 days post treatment shows relative ASPA expression normalized to actin and WT; Due to early lethality, untreated CD KO mice at p25 were used as controls (n=3 each). FIG. 26C shows brain NAA levels of treated CD KO mice quantified using magnetic resonance spectroscopy (MRS) in live mice at p42; untreated CD KO mice were used at p25 as controls. Total NAA (tNAA) relative to total creatine (tCr) is shown. Error bars indicate mean±SD; n=3; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

FIG. 27 shows the weights of the full-dose treatment groups comparing 1st, 2nd, and 3rd generation gene replacement therapy. Mice were treated at p1 with 4X1011 CG from the vectors of the three different generations. Weights were measured every other day for the first 32 days and biweekly thereafter (n=10 each). Error bars indicate mean ± SD; * p < 0.05; ** p < 0.01; ns = not significant.

FIG. 28 shows a T2 magnetic resonance imaging (MRI) sequence at p28/42. Mice treated at p1 with gene therapy from either the 1st, 2nd, or 3rd generation were subjected to MRI (n=3 per group). The T2 sequence is shown, emphasizing water-derived signals. Treated and wild-type (WT) mice were imaged at p42. Due to early lethality, untreated (knockout; KO) mice were imaged at p28. KO mice show strong hyperintensities (white signals) in the striatum, cortex, thalamus, midbrain, and cerebellum/pons. A gradual reduction of hyperintense signals is observed across treatment groups. Mice treated with the 3rd generation show the same signal pattern as WT mice, indicating a reversal of the pathological T2 MRI signals.

FIG. 29 shows that treatment of CD KO mice with 3rd generation gene therapy leads to rescue of neuropathology at p25. Mice (n=3 per group) were treated at P1 with 4X1011 CG via the facial vein and sacrificed at p25 for neuropathology. Untreated (KO) mice show extensive vacuolization throughout the brain. 1st generation treated mice show reduced vacuole formation but still have substantial defects. 3rd generation treated mice are indistinguishable from wild type (WT). Cx=cortex; St=striatum; DG=dentate gyrus; CA3=horn of Ammon 3; Thalamus=thalamus; Po=pons; Ce=cerebellum. Images are at 10x, insets are at 40x.

FIGS. 30A-30C show that optimized gene replacement therapy normalizes motor function in CD KO mice. Each trial was performed with an independent group of CD KO mice. Mice were treated at p1 with 4x10” CG into the facial vein using either 2nd or 3rd generation gene therapy (total n=24 per vector group). Test points at p27 and p365 are shown. FIG. 30A shows accelerated rotating cylinder data. FIG. 30B shows balance beam testing for balance sense deficits and ataxia. Cutoff was 300 seconds. FIG. 30C shows that muscle/grip strength was tested on an inverted screen up to 300 seconds. Error bars indicate mean ± SD; for all motor assays, n=6-8 at P27 and n=8 at p375. *** p<0.001; **** p<0.0001.

FIG. 31 shows that treatment of CD KO mice with 2nd or 3rd generation gene therapy rescues motor function at p27, p90, p180, and p365. Mice treated at p1 with 4x10” CG by facial vein delivery of 2nd or 3rd generation gene therapy were tested on the rotating cylinder, balance beam, and inverted screen over the course of 1 year. All four test time points are shown, demonstrating rescue of the motor function phenotype. Error bars indicate mean ± SD; n=6-8; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns=not significant.

FIG. 32 shows data for the T-maze at 1 year. CD KO mice treated at P1 and WT mice were tested in the T-maze for spatial/working memory and compared to untreated WT mice. Each mouse was tested 11 times with 20 seconds of retention time between each bout. Error bars indicate mean ± SD; n=6-8; ns=not significant.

FIG. 33A-33D show that optimized gene replacement therapy leads to sustained rescue of neuropathology and biomarker expression. Mice were treated at p1 intravenously with 4x10” CG of 1st, 2nd, or 3rd generation vector. Data at 1 year post-treatment are shown in comparison to wild type (WT). FIG. 33A shows MRI images comparing WT and all three vector generations. FIG. 33B shows H&E staining showing the extent of neuropathological changes. FIG. 33C shows magnetic resonance spectroscopy (MRS) analysis of total brain NAA levels normalized to total creatine at 1 year of age. FIG. 33D shows urinary NAA levels normalized to creatinine at 1 year of age quantified by mass spectrometry. Error bars indicate mean ± SD; n=3; **** p<0.0001; ns = not significant.

FIG. 34 shows that treatment of CD KO mice with 3rd generation gene therapy leads to sustained rescue of neuropathology at 1 year of age. Mice were treated with 4x10” CG of either 1st, 2nd, or 3rd generation gene therapy. Mice were sacrificed at 1 year of age and subjected to H&E staining. Images are shown at 10x with insets at 40x. Cx=cortex, St=striatum, DG=dentate gyrus, CA3=ammon’s horn 3, Thalamus=thalamus, Po=pons, Ce=cerebellum, CSC=cervical spinal cord, TSC=thoracic spinal cord, LSC=lumbar spinal cord.

FIG. 35 shows a spinning cylinder comparing wild-type versus treated wild-type mice. Mice were treated at p1 intravenously with 4X1011 CG of the 3rd generation gene therapy. Test points p27, p90, p180, and p365 are shown. Error bars indicate mean ± SD; n=8; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns=not significant.

FIG. 36A-36I show that astrocyte-restricted hASPA expression rescues motor function, neuropathology, and biomarker expression in CD KO mice. Mice were treated intravenously at p1 with 3rd generation gene therapy 4X1011 CG containing a ubiquitous or partial human glial fibrillary acidic protein (phGFAP) promoter. FIG. 36A-36B show brain sections with astrocyte-restricted EGFP expression colocalizing with GFAP-positive cells (red in H) but not myelin basic protein (MBP)-positive cells (n=3). FIG. 36C shows the weights of wild-type mice versus mice treated with ubiquitous or astrocyte-restricted hASPA expression (n=8-10). FIG. 36D shows tests of motor (accelerated rotating cylinder, balance beam, and inverted screen) and cognitive (T-maze; FIG. 36E) function from mice with astrocyte-restricted hASPA expression (n=6-8). FIG. 36F shows MRI, showing T2 signal pattern of ubiquitous versus astrocyte-restricted hASPA expression compared to wild-type and untreated mice (n=3). FIG. 36G-36H show MRS and WB of brain NAA levels and ASPA expression (n=3). FIG. 36I shows Luxol fast blue staining of myelin showing reduced myelin fibers (light arrows) and the presence of vacuoles (black arrows) in the cerebellar white matter of CD KO mice versus treated mice with ubiquitous or astrocyte-restricted hASPA expression (n=3). ML = molecular layer, PK = Purkinje cell layer, GR = granular layer, WM = white matter. Error bars indicate mean ± SD; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

FIG. 37 shows dose-dependent weights of rAAV. Weights per day (inset) and week of CD KO mice treated at p1 with either a full, 3-fold, or 10-fold lower dose of the 3rd generation gene therapy construct are shown. For comparison, CD KO mice were treated with the 1st generation gene therapy at a 3-fold lower dose. Mice treated with a 10-fold lower dose are only shown for the first four weeks. For weights per week of CD KO mice treated with the full 3rd generation dose, see Fig. S1. Error bars indicate mean ± SD; n=8-10, except for 0.3X 1st gen. after 24 weeks because animals began to die; **** p<0.0001.

FIGS. 38A-38D show that dose reduction of 3rd generation therapy achieves effective disease rescue in CD KO mice. CD KO mice were treated at p1 via the facial vein with 4x1010, 1.33x10”, or 4x10” CG of 3rd generation gene replacement therapy. FIG. 38A shows T2 MRI sequences of different brain regions with wild type (WT) and untreated (KO) control mice. All mice were imaged at p25 (n=3). FIG. 38B shows total NAA levels from the same mice as in FIG. 38A quantified by MRS at p25 and normalized to total creatine (n=3). FIG. 38C shows dose-dependent neuropathology by H&E of p25 mice treated with 3rd generation vectors or full dose of 1st generation. WT and KO (n=3) were used as controls. FIG. 38D shows dose-dependent assessment of motor function on the rotating cylinder, balance beam, and inverted screen at p27 and p90 (n=6-8). Error bars indicate mean ± SD; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

FIG. 39 shows dose-dependent neuropathology. CD KO pups were treated intravenously at p1 with 4x10”, 1.33x10”, or 4x1010 3rd generation gene therapy CG. Mice were sacrificed at p25 and subjected to H&E staining and microscopy. 10X magnifications of 7 different brain regions are shown. Wild-type (WT) and untreated (KO) mice were used as controls. See legend in FIG. 29.

FIG. 40 shows that rAAV vector genome copy number varies between brain regions of treated CD KO mice. Eleven different regions of the CNS were analyzed for rAAV9 vector genome copy number per cell. A schematic drawing of a sagittal section of the mouse brain is shown with 11 brain regions, including the spinal cord, labeled and highlighted. Vector genome copy number per diploid cell (rAAV genome/cell) is shown for WT control, and 1-year-old CD KO mice treated with full dose of 1st generation, and full and 3-fold lower dose of 3rd generation gene therapy. On the left, a classification of the rAAV genome/cell of the treated 3rd generation is shown. CBL = cerebellum, LMN = quadrigeminal plate, MB = midbrain, OB = olfactory bulb, LSC = lumbar spinal cord, TSC = thoracic spinal cord, BS = brainstem, CSC = cervical spinal cord, TAL = thalamus and hypothalamus, HPC = hippocampus, Cx = cortex. Error bars indicate mean ± SD; n = 3-4; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.

FIG. 41 shows Luxol fast blue staining and astrocytic expression of ASPA. CD KO mice were treated at p1 via the facial vein with 4x10” 3rd generation gene therapy CG or GFAPhASPAOpt in rAAV9. 5 brain regions from p25 mice are shown versus wild type (WT) and untreated control (KO) mice. 10x magnifications are shown. See legend in FIG. 29.

FIGS. 42A-42F show high-field imaging to noninvasively assess therapy outcomes. Male mice were treated at p1 and imaged at p25, comparing untreated CD KO mice (UT), wild-type mice (WT), and treated CD KO mice (Tx) (n=8-10 each). FIG. 42A shows representative tractography images of the corpus callosum. FIG. 42B shows representative images of fractional anisotropy (FA) values of the left and right external capsule (EC) and corpus callosum (CC). FIG. 42C shows 19 brain regions selected for analysis of resting-state functional magnetic resonance imaging (RS-fMRI) results and mapped onto a sagittal brain map. Light and black dots indicate cortical and subcortical brain regions, respectively. Light lines show all connections involving cortical regions and darker lines show only subcortical connections. FIG. 42D shows ER-fMRI T-score statistics indicating differences between corresponding brain regions and overall brain activity (shown in FIG. 42C). FIG. 42E shows total N-acetylaspartate (NAA) normalized against total creatine (tCr) that was measured in all mice that underwent DTI and ER-fMRI imaging. FIG. 42F shows that overall functional connectivity (31 untreated, 21 wild-type, 19 treated) was correlated with mean accelerated rotating cylinder performance in seconds (FIG. 33A, p27) and was analyzed by linear regression analysis (R2 = 0.89). Error bars indicate mean ± SD; n=8-10; p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns = not significant.

FIG. 43 shows the resting-state functional magnetic resonance imaging (RS-fMRI) regions of interest. Nineteen regions of interest (ROIs) were selected to represent regions of motor and cognitive function. N=9-10 mice per group were imaged while anesthetized to acquire RS-fMRI.

FIG. 44 shows that NAA levels correlate with T2 signal intensities.

FIG. 45 shows myelin restoration 4 weeks after treatment with the hASPA construct.

FIG. 46 shows data from the rotating cylinder assay in male mice. The data indicate an expanded therapeutic window for ASPA gene therapy. Mice were tested at 4 weeks, 10 weeks, 16 weeks, 28 weeks, and 52 weeks.

FIG. 47 shows data from the rotating cylinder assay in female mice. The data indicate an expanded therapeutic window for ASPA gene therapy. Mice were tested at 4 weeks, 10 weeks, 16 weeks, 28 weeks, and 52 weeks.

FIG. 48 shows T-maze data indicating that working/spatial memory is restored in Nur7 mice following treatment with the 3rd generation hASPA gene therapy construct. Cognitive function of Nur7 mice treated at neonatal, juvenile, or adult ages was assessed at 1 year of age.

FIG. 49 shows gait analysis data indicating a therapeutic benefit for mice treated with 3rd generation ASPA gene therapy at 6 months and earlier.

FIG. 50 shows gait analysis data indicating that mice treated at mature adulthood (p168) continue to benefit from 3rd generation ASPA gene therapy treatment.

FIG. 51 shows mitochondrial stress test data comparing ASPA-deficient HEK cells to wild-type HEK cells (e.g., non-ASPA-deficient cells).

FIG. 52 shows mitochondrial profiling data from ASPA-deficient HEK cells (HEK293 KO) compared to wild-type HEK cells (HEK293 WT).

FIG. 53 shows data indicating that ASPA-deficient cells use more fatty acids and glutamine for energy production. Data are shown regarding the dependence, flexibility, and ability of wild-type or ASPA-deficient cells to use glucose (GLC), glutamine (GLN), or fatty acids (FA) for energy production.

FIG. 54 shows data indicating that Nur7 mice treated with the 3rd generation ASPA gene therapy construct have a significantly lower g ratio than untreated Nur7 mice, indicating increased myelin thickness due to remyelination.

FIG. 55 shows data relating to the measurement of beta-oxidation in untreated ASPA-deficient mice, wild-type mice, treated control mice, and mice treated with the 3rd generation ASPA gene therapy construct.

FIG. 56 shows the decrease in glycolysis in ASPA-deficient brain and its restoration after ASPA reconstitution.

FIG. 57 shows additional representative data from whole-brain metabolome analysis in wild-type (WT) and ASPA knockout (KO) mice.

DETAILED DESCRIPTION

Aspects of the disclosure relate to methods of treating neurodegenerative diseases (e.g., leukodystrophies) in a subject in need thereof. The methods provided herein involve modulating N-acetylaspartate (NAA) levels in a subject. NAA has been identified as the second most abundant molecule in the central nervous system (CNS). NAA synthesis can occur in neurons. In some cases, NAA is not synthesized in cells or organs outside of the CNS. NAA is metabolized by the enzyme aspartoacylase (ASPA) into acetate and L-aspartate. ASPA can be expressed in the CNS (e.g., in oligodendrocytes). ASPA can be expressed in peripheral organs, such as the kidney, small intestine, and others. Neurodegenerative diseases may show alterations in NAA metabolism. Consequently, NAA can be used as a disease marker for a wide range of CNS disorders, e.g., Canavan disease, Alzheimer's disease, traumatic brain injury, and psychiatric disorders. Other examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease, traumatic brain injury (TBI), bipolar disorder, catalepsy, epilepsy (e.g., seizures), migraine, Huntington's disease, attention deficit disorder (ADD), attention deficit hyperactivity disorder (e.g., ADHD), autism spectrum disorder (e.g., Asperger's disease, autism, etc.), Parkinson's disease, Tourette syndrome, clinical depression, multiple sclerosis, and autoimmune disease (e.g., CNS demyelinating disease, myasthenia gravis, etc.).

Methods are provided for treating leukodystrophy in a subject in need thereof, which involve administering to the subject an N-acetylaspartate (NAA)-reducing agent. As used herein, the term "NAA-reducing agent" refers to an agent (e.g., nucleic acid, protein, small molecule) that reduces NAA levels directly or indirectly. Leukodystrophy may have been determined to be associated with a metabolic imbalance comprising a shift from glycolysis to beta-oxidation in the subject.

Other aspects of the disclosure relate to methods of treating neurodegenerative disease in a subject in need thereof, wherein the methods involve administering to the subject an N-acetylaspartate (NAA)-increasing agent. As used herein, the term "NAA-increasing agent" refers to an agent (e.g., nucleic acid, protein, small molecule) that increases NAA levels directly or indirectly. In some embodiments, the neurodegenerative disease has been determined to be associated with a metabolic imbalance comprising a NAA deficiency, e.g., by administering siRNA/shRNA or miRNA, e.g., that inhibit ASPA expression. As used herein, "metabolic imbalance" refers to a dysregulated or abnormal metabolic state in a subject. For example, the CNS cells of a healthy subject may exhibit a preference for glycolysis as the primary mode of energy (e.g., ATP production); In subjects suffering from certain neurodegenerative diseases (e.g., diseases associated with leucodystrophy, e.g., Canavan disease), CNS cells may show a preference for fatty acid metabolism. Such a shift away from glycolysis and toward beta-oxidation may be termed "metabolic imbalance."

The methods disclosed herein may involve comparing biomarkers (e.g., beta-oxidation, glycolysis) to an appropriate control. An “appropriate control” refers to a level of a particular biomarker (e.g., beta-oxidation, glycolysis) that is indicative of a known metabolic state. Such levels may be determined experimentally or may be pre-existing reference levels. In some embodiments, an appropriate control may be a biomarker level indicative of the presence of a metabolic imbalance. For example, an appropriate control may be the level of a factor (e.g., beta-oxidation, glycolysis) in a control subject. A control subject may not have a metabolic imbalance. However, a control subject may have a metabolic imbalance.

Recombinant adeno-associated viruses (rAAV)

In some aspects, the disclosure provides isolated AAVs that are useful for delivering transgenes encoding NAA modulating agents (e.g., an NAA reducing agent, an NAA increasing agent). As used herein with respect to AAVs, the term "isolated" refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as "recombinant AAVs." Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities, such that a nuclease and/or transgene of the rAAV will be specifically delivered to one or more predetermined tissues. The capsid of the AAV is an important element in determining this tissue-specific targeting. Thus, a rAAV may be selected that has a capsid suitable for the tissue being targeted.

In some aspects, the disclosure provides an rAAV having a capsid suitable for targeting central nervous system (CNS) tissue or other tissue (e.g., a peripheral tissue). The capsid may have a serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, and AAVrh.10. An rAAV may comprise variants of the capsid proteins of serotypes AAV1, AAV2, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, and AAVrh.10. The capsid proteins may comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any of the recited capsids.

Suitable methods can be used to obtain recombinant AAVs having a desired capsid protein. (See, e.g., US Patent No. 2003/0138772.) Typically, the methods involve culturing a host cell containing a nucleic acid sequence encoding an AAV capsid protein; a functional gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to allow encapsidation of the recombinant AAV vector into the AAV capsid proteins. The capsid proteins may be structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 through 3 (referred to as VP1, VP2, and VP3), all of which are transcribed from a single cap gene by alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are, respectively, about 87 kDa, about 72 kDa, and about 62 kDa. Upon translation, the capsid proteins may form a 60-mer spherical protein shell around the viral genome. The functions of the capsid proteins may be to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid proteins deliver the viral genome to a host in a tissue-specific manner.

The components that must be grown in the host cell to encapsidate a rAAV vector into an AAV capsid may be provided to the host cell entrans. Alternatively, one or more of the required components (e.g., the recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell that has been manipulated to contain one or more of the required components using methods known to those skilled in the art. Most suitably, such a stable host cell contains the necessary component(s) under the control of an inducible promoter. However, the necessary component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of suitable regulatory elements for use with the transgene. In another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and another selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated that is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Other stable host cells may be generated by one of skill in the art.

The present disclosure relates to a host cell containing a nucleic acid comprising a coding sequence encoding a gene associated with a neurodegenerative disease (e.g., a leukodystrophy). The present disclosure may relate to a composition comprising the host cell described above. The composition comprising the above host cell may further comprise a cryopreservative.

The recombinant AAV vector, rep sequences, cap sequences, and helper functions necessary to produce the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. Methods used to construct any embodiment of the present disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Likewise, methods of generating rAAV virions are well known, and selection of a suitable method is not a limitation of the present disclosure. See, for example, K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

Recombinant AAVs can be produced using the triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) that will be encapsidated into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap), which function to transduce productive AAV replication and encapsidation. Preferably, the AAV helper function vector allows for efficient production of AAV vectors without generating detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Patent No. 6,001,650 and the pRep6cap6 vector, described in U.S. Patent No. 6,156,303. The accessory function vector encodes nucleotide sequences for non-AAV-derived viral and/or cellular functions upon which AAV depends for its replication (i.e., “accessory functions”). Accessory functions include those functions required for AAV replication, including, without limitation, those residues involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Accessory virus-based functions may be derived from any of the known helper viruses, such as adenoviruses, herpesviruses (other than herpes simplex virus type 1), and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced into the interior of the cell membrane. In general, various transfection techniques are known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al., (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integrating vector and other nucleic acid molecules, into suitable host cells.

A "host cell" refers to any cell that harbors, or is capable of harboring, a substance of interest. Often, a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell that has been transfected. Thus, a "host cell," as used herein, may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.

As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Cell lines are often clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes in karyotype may occur during storage or transfer of such clonal populations. Therefore, cells derived from the referenced cell line may not be exactly identical to the ancestral cells or cultures, and the referenced cell line includes such variants.

As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that leads to the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as an RNA.

As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., capable of replication when associated with appropriate control elements and capable of transferring gene sequences between cells. Thus, the term includes cloning and expression vehicles as well as viral vectors. Useful vectors are those in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the cell's synthetic machinery, or introduced synthetic machinery, necessary to initiate specific transcription of a gene. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct location and orientation relative to the nucleic acid to control initiation of RNA polymerase and expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid into which part or all of the coding sequence of the nucleic acid can be transcribed. Expression may include transcription of the nucleic acid, for example, to generate a biologically active polypeptide product or functional RNA (e.g., shRNA, miRNA) from a transcribed gene.

The above methods for encapsidating recombinant vectors into desired AAV capsids to produce the rAAVs of the disclosure are not intended to be limiting and other suitable methods will be apparent to the skilled artisan.

Isolated nucleic acids

A “nucleic acid” sequence refers to a DNA or RNA sequence. Proteins and nucleic acids of the disclosure may be isolated. As used herein, the term “isolated” means artificially produced. As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, the polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, such as by excision and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one that is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which the 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated, but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but is not necessarily so. For example, an isolated nucleic acid that is isolated within a cloning or expression vector is not pure, as it may comprise only a small percentage of the material of the cell in which it resides. However, such a nucleic acid is isolated, as the term is used herein, because it is readily manipulable by standard techniques known to those skilled in the art. As used herein with respect to proteins or peptides, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or produced artificially (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

Conservative amino acid substitutions may be made to provide functionally equivalent variants or homologs of the capsid proteins. In some aspects, the disclosure encompasses sequence alterations that result in conservative amino acid substitutions. As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be prepared according to methods for altering the polypeptide sequence known to one of skill in the art, such as those found in references compiling such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus, conservative amino acid substitutions can be made in the amino acid sequence of the proteins and polypeptides disclosed herein.

Recombinant AAV vectors (rAAV vectors)

"Recombinant AAV (rAAV) vectors" of the disclosure are typically comprised of, at a minimum, a transgene and its regulatory sequences, and AAV 5' and 3' inverted terminal repeats (ITRs). It is this recombinant AAV vector that is encapsidated in a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, that encodes a polypeptide, protein, functional RNA molecule (e.g., shRNA, miRNA), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and/or expression of the transgene in a cell of a target tissue.

In some embodiments, the present disclosure relates to a recombinant AAV (rAAV) vector comprising a nucleic acid sequence that includes a promoter operably linked to a transgene, wherein the transgene is a gene associated with a neurodegenerative disease (e.g., leukodystrophy). A rAAV vector may further comprise nucleic acid sequences encoding one or more AAV inverted terminal repeat (ITR) sequences, e.g., AAV2 ITRs. A rAAV vector may further comprise nucleic acid sequences encoding one or more AAV ITRs selected from the group consisting of AAV2, AAV3, AAV4, AAV5, and AAV6.

AAV vector sequences typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (see, for example, B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially the complete ITR-encoding sequences are used in the molecule, although some degree of minor modification of these sequences is permitted. The ability to modify these ITR sequences is within the skill of the art. (See, for example, texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520-532 (1996)). An example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing the transgene, wherein the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types (e.g., AAV2, AAV3, AAV4, AAV5, or AAV6 ITR sequences).

The rAAVs of the present disclosure may be pseudotyped rAAVs. Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped viral particle. With this method, foreign viral envelope proteins may be used to alter host tropism or to increase/decrease the stability of the virus particles. In some aspects, a pseudotyped rAAV comprises nucleic acids from two or more different AAVs, wherein the nucleic acid of one AAV encodes a capsid protein and the nucleic acid of at least one other AAV encodes other viral proteins and/or the viral genome. A pseudotyped rAAV may refer to an AAV that comprises an inverted terminal repeat (ITR) from one AAV serotype and a capsid protein from a different AAV serotype. For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y would be designated AAVX/Y (e.g., AAV2/1 has the ITRs of AAV2 and the capsid of AAV1). Pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with viral DNA from another AAV serotype, thus allowing for targeted delivery of a transgene to a target tissue.

In addition to the core elements identified above for the recombinant AAV vector, the vector also includes necessary control elements that are operably linked to the transgene in a manner that allows its transcription, translation, and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. A large number of expression control sequences are known in the art, including native, constitutive, inducible, ubiquitous, and/or tissue-specific promoters that may be used.

As used herein, a nucleic acid sequence (e.g., a coding sequence) and regulatory sequences are said to be "operably" linked when they are covalently linked such that expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter at the 5' regulatory sequences results in transcription of the coding sequence and if the nature of the junction between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript could be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that have been translated in frame. Operably linked coding sequences can give rise to a fusion protein. Operably linked coding sequences can give a functional RNA (e.g., shRNA).

For nucleic acids encoding proteins, a polyadenylation sequence is generally inserted following the transgene sequences and before the AAV 3' ITR sequence. An rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein containing more than one polypeptide chain. Selection of these and/or other vector elements can be made as appropriate, and many such sequences are available [see, for example, Sambrook et al., and references cited therein at, for example, pages 3,183,26 and 16,17 16,27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, a foot-and-mouth disease virus 2A sequence is included in the polyprotein; It is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, Nov 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). Cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4:928-933; Mattion, N M et al., J Virology, Nov 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6:198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11:1921-1931.; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

The precise nature of the regulatory sequences necessary for gene expression in the host cells may vary among species, tissues, or cell types, but will generally include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like. Especially, such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably linked gene. The regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired. The vectors of the disclosure may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one skilled in the art.

Examples of constitutive promoters include, but are not limited to, the Rous sarcoma virus (RSV) retroviral LTR promoter (optionally with RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen]. In some embodiments, a promoter is an improved chicken p-actin promoter. In some embodiments, a promoter is an astrocyte-specific promoter. In some embodiments, a promoter is an oligodendrocyte-specific promoter. In some embodiments, a promoter is a CNS-specific promoter.

Inducible promoters allow for regulation of gene expression and may be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have been described that can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the insect ecdysone promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters that may be useful in this context are those that are regulated by a specific physiological state, for example, temperature, the acute phase, a particular differentiation state of the cell, or only in replicating cells.

The native promoter of the transgene may be used. The native promoter may be preferred when it is desired that the expression of the transgene mimic native expression. The native promoter may be used when the expression of the transgene must be regulated temporally or evolutionarily, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In another embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.

Regulatory sequences may confer tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Exemplary tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: a liver-specific thyroxine-binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an α-myosin heavy chain (α-MHC) promoter, or a cardiac troponin T (cTnT) promoter. Other exemplary promoters include the beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); the immunoglobulin heavy chain promoter; the T-cell receptor alpha chain promoter, neuronal such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)) and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others that will be apparent to the expert. The promoter may be an oligodendrocyte-specific promoter, for example, the myelin basic protein (MBP) promoter (Chen et al., J. Neurosci, Res., 55(4); 504-13 (1999)).

Aspects of the disclosure relate to the discovery that astrocyte-specific (e.g., astrocyte-restricted) expression of hASPA has a positive therapeutic effect (e.g., survival, normalized growth, restoration of normal motor function and cognitive function) in mouse models of Canavan disease. Thus, in some embodiments, the rAAV transgene described by the disclosure is operably linked to an astrocyte-specific promoter. Examples of astrocyte-specific promoters include, but are not limited to, the glial fibrillary acidic protein (GFAP) promoter (Brenner et al., J. Neurosci, 14(3, Pt 1); 1030-7 (1994)), the aldehyde dehydrogenase family 1, member L1 (ALDH1L1) promoter (Cahoy et al., J. Neurosci. 28, 264-278 (2008)), and the glutamate transporter EAAT1 promoter (Colin et al., Glia 57, 667-679 (2009)). In some embodiments, the astrocyte-specific promoter is the glial fibrillary acidic protein (GFAP) promoter.

One or more binding sites for one or more miRNAs may be incorporated into a transgene of a rAAV vector, to inhibit expression of the transgene in one or more tissues of a subject harboring the transgene. It will be appreciated by those skilled in the art that binding sites may be selected to control expression of a transgene in a tissue-specific manner. For example, binding sites for the liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. Target sites in the mRNA may be in the 5' UTR, the 3' UTR, or in the coding region. Typically, the target site is located in the 3' UTR of the mRNA. Furthermore, the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may result in the cooperative action of multiple RISCs and provide highly effective inhibition of expression. The target site sequence may comprise a total of 5-100, 10-60, or more nucleotides. The target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site.

Recombinant AAV Administration Methods

rAAVs may be administered to a subject in compositions by any appropriate method. The rAAV, for example, suspended in a physiologically compatible carrier (i.e., in a composition), may be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some cases, a host animal does not include a human.

Compositions of the disclosure may comprise a rAAV alone, or in combination with one or more viruses (e.g., a second rAAV having one or more different transgenes). A composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

In some cases, administration of a rAAV to a subject elicits an immune response against the rAAV capsid protein in the subject. Without wishing to be bound by any particular theory, suppression of a subject's immune system prior to administration of a rAAV results, in some embodiments, in an enhanced therapeutic effect of the rAAV. Thus, one or more (e.g., 2, 3, 4, 5, or more) immunosuppressive agents may be administered to a subject prior to administration of a rAAV as described in the disclosure. An "immunosuppressive agent" is any composition (e.g., a protein, nucleic acid, small molecule, etc.) that reduces a subject's immune response to a rAAV. An immunosuppressive agent may reduce the innate immune response, the adaptive immune response, the cellular immune response, the humoral immune response, or any combination of the above, in a subject.

Examples of biological immunosuppressive agents include, but are not limited to, monoclonal antibodies, such as monoclonal antibodies that block the costimulatory pathway (e.g., appropriate antibodies against CTLA4, ICOS, CD80, OX40, or other targets), interfering RNA (e.g., siRNA, dsRNA, shRNA, miRNA, etc.) targeting immunostimulatory molecules (e.g., cytokines), and proteins (e.g., proteasome inhibitors).

Examples of small molecule immunosuppressive molecules include, but are not limited to, glucocorticoids (e.g., cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone, deoxycorticosterone (DOCA), and aldosterone), cytostatics (e.g., cyclophosphamide, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, etc.), drugs targeting immunophilins (e.g., cyclosporine, tacrolimus, sirolimus, rapamycin, etc.), interferons (e.g., IFN-p), mycophenolate, fingolimod and myriocin.

An immunosuppressive agent may be administered to a subject between about one week and one minute prior to administration of a rAAV as described herein. In some embodiments, an immunosuppressive agent is administered to a subject between about 5 days, about 1 day, about 12 hours, about 2 hours, about 1 hour, about 30 minutes, about 10 minutes, about 5 minutes, or about 1 minute prior to administration of a rAAV. An immunosuppressive agent may be administered to a subject on multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) occasions prior to administration of a rAAV to the subject.

A composition may further comprise a pharmaceutically acceptable carrier. Suitable carriers can be readily selected by one of skill in the art in view of the indication for which the rAAV is intended. For example, a suitable carrier includes saline, which can be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of carrier is not a limitation of the present disclosure.

Optionally, the compositions of the disclosure may contain, in addition to the rAAV and the carrier(s), other pharmaceutical components, such as preservatives or chemical stabilizers. Some suitable exemplary preservatives are chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

rAAVs are administered in amounts sufficient to transfect cells of a desired tissue (e.g., CNS tissue) and provide sufficient levels of gene transfer and expression without undue adverse effects. Examples of pharmaceutically acceptable routes of administration include, but are not limited to, direct administration to the selected organ (e.g., intrathecal, intracerebral), oral, inhalation (including intranasal and intratracheal administration), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. Routes of administration may be combined, if desired.

The dose of rAAV virions required to achieve a particular "therapeutic effect", e.g., dosage units in genomic copies/per kilogram of body weight (CG/kg), will vary based on several factors, including, but not limited to: the route of administration of the rAAV virion, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a dosage range of rAAV virions to treat a patient with a particular disease or disorder based on the factors discussed above, as well as other factors.

An effective amount of a rAAV is an amount sufficient to infect an animal and a desired tissue. An effective amount of a rAAV may be an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue being targeted, and may vary by animal and tissue. For example, an effective amount of rAAV is generally in the range of about 1 mL to about 100 mL of solution containing about 10 to 10 genome copies (cg). A dose between about 10 and 10 genome copies may be suitable. In some cases, a dose between about 10 and 10 rAAV genome copies is suitable. In certain cases, 10 or 10 rAAV genome copies is effective for targeting CNS tissue. The dose of an rAAV may be calculated based on the weight of the subject to which the rAAV is administered. For example, a dose between 1.0 x 1010 cg/kg and 1.0 x 1015 cg/kg may be appropriate. A dose of 2.0 x 1010 cg/kg, 2.0 x 1011 cg/kg, 2.0 x 1012 cg/kg, 2.0 x 1013 cg/kg, 2.0 x 1014 cg/kg, or 2.0 x 1015 cg/kg may be appropriate. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

A dose of rAAV may be administered to a subject no more than once per calendar day (e.g., in a 24-hour period). A dose of rAAV may be administered to a subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. A dose of rAAV may be administered to a subject no more than once per calendar week (e.g., 7 calendar days). A dose of rAAV may be administered to a subject no more than twice per week (e.g., once in a two-calendar-week period). A dose of rAAV may be administered to a subject no more than once per calendar month (e.g., once every 30 calendar days). A dose of rAAV may be administered to a subject no more than once every six calendar months. A dose of rAAV may be administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

rAAV compositions may be formulated to reduce aggregation of AAV particles in the composition, particularly when high concentrations of rAAV are present (e.g., ~1013 CG/ml or greater). Appropriate methods may be used to reduce aggregation, including, for example, addition of surfactants, pH adjustment, adjustment of salt concentration, etc. (see, for example, Wright FR, et al., Molecular Therapy (2005) 12, 171-178).

The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared in such a manner that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, as well as other pharmacological considerations, will be contemplated by one skilled in the art in the preparation of such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

The rAAVs in the suitably formulated pharmaceutical compositions disclosed herein may be administered directly to the target tissue, for example, directly to the CNS tissue. However, under certain circumstances it may be desirable to separately or additionally administer the rAAV-based therapeutic constructs by another route, for example, subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally or orally, intraperitoneally, or by inhalation. The administration modalities described in U.S. Patent Nos. 5,543,158; 5,641,515, and 5,399,363 may be used to administer rAAVs.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols and

mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that ready syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Adequate fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption in the compositions, for example, aluminum monostearate and gelatin.

For the administration of an aqueous injectable solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a suitable sterile aqueous medium may be employed. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclysis fluid or injected into the proposed site of infusion (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. In any case, the person responsible for the administration will determine the appropriate dosage for each host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other components listed herein as required, followed by sterile filtration. In general, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing the basic dispersion medium and the necessary other components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques which produce a powder of the active ingredient plus any desired additional components from a previously sterile filtered solution thereof.

The rAAV compositions disclosed herein may also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Once formulated, the solutions will be administered in a manner compatible with the formulation and in the therapeutically effective amount. The formulations are readily administered in various dosage forms, such as injectable solutions, drug-releasing capsules, and the like.

As used herein, the term "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients may also be incorporated into the compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

Delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for introduction of the compositions of the present disclosure into suitable host cells. In particular, transgenes delivered by the rAAV vector may be formulated for administration encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere or a nanoparticle, or the like.

Such formulations may be preferable for the introduction of pharmaceutically acceptable formulations of the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-times have been developed (U.S. Patent No. 5,741,516). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully in a variety of cell types that are normally resistant to transfection by other methods. In addition, liposomes do not have the DNA length limitations typical of viral-based delivery systems. Liposomes have been used effectively to deliver genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several clinical trials examining the efficacy of liposome-mediated drug delivery have been successfully completed.

Liposomes consist of phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have diameters ranging from 25 nm to 4 gm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 ANG., which contain an aqueous solution in the core.

Alternatively, nanocapsule formulations of rAAV can be used. Nanocapsules can generally entrap substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (around 0.1 pm in size) should be designed using polymers capable of in vivo degradation. The use of biodegradable poly(alkyl cyanoacrylate) nanoparticles that meet these requirements is envisaged.

EXAMPLES

Example 1: Pathomechanism of Canavan disease

Experimental design

Global biochemical profiles were determined in brain tissue from mice collected from postnatal day 25 (P25) representing the treatment groups shown below in Table 1.

Table 1: Treatment groups

Metabolomics of healthy mouse brains and those with Canavan disease

Resting-state functional MRI (RS-fMRI) results indicate that Canavan disease causes increased oxygen consumption for functional neuroconnectivity (Fig. 2). Furthermore, Canavan disease causes increased oxygen consumption for cortical/subcortical connectivity (Fig. 3). These data indicate that Canavan disease may be characterized by an altered metabolic state in the CNS.

The molecular phenotype of brains from Canavan disease mice was investigated using a whole-brain metabolomic approach. Canavan disease was treated with intravenous (IV) injection of rAAV-ASPA at p1. Brains from healthy and CD mice (treated and untreated groups) were homogenized and subjected to metabolic analysis. More than 452 metabolites were quantified in each wild-type, untreated, treated, and treatment control group (Table 2). This large dataset revealed several crucial and entirely new aspects about the pathomechanism of Canavan disease, its gene therapy, CNS metabolism, and the novel function of AspA in general.

Table 2: Results of metabolomic analysis

Statistical analysis

Principal component analysis (PCA) transforms a large number of metabolic variables into a smaller number of orthogonal variables (Component 1, Component 2, etc.) to analyze variation between groups and provide a high-level overview of the dataset. In PCA (FIG. 4A), the samples were formed into two populations; interestingly, rather than reflecting the gene pool, these two populations appeared to reflect disease status: WT and rAAV Tx formed the left population, while KO and rAAV Ctrl formed the right population, consistent with a “rescue” of the disease by treatment.

Hierarchical clustering analysis (HCA) examines similarities between groups (Fig. 4B); consistent with PCA observations, the top-level separation of the dendrogram distinguished between WT and rAAV Tx samples (left) and KO and rAAV samples (right), with subclusters consisting of individual samples per group. Notably, both PCA and HCA clustered WT and rAAV Tx samples together. A high-level assessment of the patterns of metabolic changes in HCA indicates that WT and rAAV Tx tended to show similar trends in a number of metabolites, indicating that rAAV Tx was effective in modulating disease. Many of the changes observed in KO (compared to WT) trended in the opposite direction in rAAV (Tx vs. Ctrl), consistent with “rescue” of disease-associated phenotypes. Consistent with the metabolic analysis, FIG. 1 shows that rAAV-ASPA gene therapy restores the thalamocortical tract of CD mice, as measured by diffusion tensor MRI for cerebral water flow.

Biosynthesis of neurotransmitters

Aspartoacylase (ASPA) is responsible for the breakdown of N-acetylaspartate (yielding acetate and aspartate). Consistently, N-acetylaspartate (NAA) was elevated in KO (compared to WT); treatment with rAAV-expressing ASPA resulted in decreased NAA (and increased aspartate). Interestingly, while NAA levels were increased (KO vs WT), the neuropeptide N-acetyl-aspartyl-glutamate (NAAG) was not significantly changed (which could reflect changes in demand or steady-state pool regulation/pool size). Gamma-aminobutyrate (GABA) was decreased (KO vs WT), which could reflect changes in GABA-mediated signaling (GABA was increased in rAAV Tx compared to rAAV Ctrl).

Other neurotransmitters were also detected in the dataset; while acetylcholine and serotonin did not undergo significant changes in KO (compared to WT), serotonin did show increases in rAAV Tx (compared to Ctrl), which could reflect changes in serotonergic signaling.

Glucose metabolism

In the absence of disease, energy in the brain is thought to be focused on glycolytic use, with acetyl CoA entering the TCA cycle to support oxidative metabolism and macromolecule biosynthesis. Increases in glucose, glucose 6-phosphate, and an isobar of sugar diphosphates (fructose 1,6-diphosphate, glucose 1,6-diphosphate, myo-inositol 1,4- or 1,3-diphosphate) could indicate changes in glucose use or increased availability.

Glucose and related molecules (fructose, mannose, and myo-inositol) were elevated, although nucleotide sugars (e.g., UDP-glucose, UDP-galactose) decreased, which could indicate a shift in biosynthetic demand (KO vs. WT). The three-carbon glycolytic intermediates, 3-phosphoglycerate (3-PG) and phosphoenolpyruvate (PEP), also increased; stores of these biochemicals tend to increase as glycolytic use decreases.

Consistent with decreased glycolytic use, lactate decreased (with a nonsignificant decrease in pyruvate). Glycogen metabolites (maltotetraose, maltotriose, and maltose) also increased, reflecting decreased glycolytic use. The energetic changes reflect decreased energy demand (potentially associated with increased neuronal cell death or senescence) or may reflect metabolic effects of NAA accumulation in the brain.

In rAAV (Tx vs Ctrl), decreased glucose and related molecules, with increases in lactate, were indicative of increased glycolytic utilization. Interestingly, DHAP was elevated in rAAV (Tx vs Ctrl), which could reflect a shift in utilization for triglyceride biosynthesis (potentially related to a restoration of lipid biosynthesis, TAG may be used as a phospholipid precursor). Representative data referring to glucose metabolism are provided in FIG. 5.

Lipid metabolism

Complex lipids, sphingolipids, diacylglycerols, monoacylglycerols, and plasmalogens were decreased, as were lysolipids, long-chain fatty acids (e.g., palmitate, palmitoleate, and stearate) and polyunsaturated fatty acids, and longer acylcarnitines (e.g., myristoylcarnitine, palmitoylcarnitine) indicative of a shift in availability or use to support beta-oxidation (KO vs. WT). The ketone body 3-hydroxybutyrate (BHBA) was also elevated in KO (compared to WT), with decreases in malonylcarnitine (a surrogate for malonyl CoA) indicative of a shift toward greater fatty acid beta-oxidation. Increases in BHBA may also reflect changes in hepatic ketogenesis (or increased brain ketone uptake to supplement energy).

Increases in carnitine, deoxycarnitine, and changes in coenzyme A precursors (increases in pantothenate with decreases in 3'-dephosphocoenzyme A and coenzyme A) could reflect changes in demand or use (KO vs. WT). N-acetylaspartate has been suggested to be a key transporter of 2-carbon units into oligodendrocytes for lipid biosynthesis; decreased lipids could reflect increased demand related to decreased biosynthesis.

Interestingly, in rAAV (Tx vs Ctrl), increases in malonylcarnitine might imply shifts towards increased fatty acid biosynthesis; metabolites related to phospholipid biosynthesis and remodeling (e.g., choline, CDP-choline, phosphoethanolamine) were also elevated. Finally, decreases in sphingolipids (e.g., sphinganine, sphingosine, and sphingomyelins) in KO (compared to WT), with increases in serine and threonine, might reflect a shift in availability for myelin biosynthesis, which has been suggested as one of the causes of neuronal cell death in Canavan disease; rAAV (Tx vs Ctrl) showed increases in these biochemicals. Representative data relating to lipid metabolism are provided in FIG. 6.

Redox homeostasis

Changes in metabolites related to glutathione biosynthesis (e.g., methionine, cystathionine, and cysteine) might indicate alterations in redox homeostasis in KO (compared to WT) (FIG. 7). Glutathione (oxidized or reduced) was decreased, as were related oxidized products (S-methylglutathione and S-lactoylglutathione), likely reflecting decreased glutathione availability. Finally, gamma-glutamyl amino acids tended to be decreased as a class (potentially reflecting decreased glutathione and/or amino acid availability); the decrease in 5-oxoproline might indicate decreased exchange of gamma-glutamyl amino acids to regenerate glutathione. The changes in KO (compared to WT) were indicative of a less robust redox environment; However, no significant differences were observed in oxidized lipids (e.g., 4-hydroxy-nonenalglutathione, 9/13-HODE) and methionine or cysteine oxidation products (methionine sulfoxide, cysteine sulfinic acid). Given the overall decrease in parental metabolites, the “similar” levels in KO compared to WT may reflect a relative increase in these products (as a result of increased oxidative stress). Changes in endogenous antioxidants, such as decreases in vitamin C metabolites (ascorbate, dehydroascorbate, and threonate) and dipeptide products of histidine with antioxidant function (anserine (FIG. 7), homocarnosine), and increases in taurine and N-acetyltaurine, could reflect use to balance changes in redox homeostasis. Finally, changes in rAAV (Tx vs. Ctrl) were consistent with decreased oxidative stress (showing essentially inverse changes to those observed in KO vs. WT).

Gene therapy in Canavan disease

Although gene therapy in patients with CD using intraparenchymal injections of ASPA expression systems was considered safe, it failed to show clinically significant improvements. Similar results were found using acetate substitution.

The data described herein demonstrate that intraventricular (e.g., direct injection) or intravenous (e.g., systemic) injection of recombinant adeno-associated virus (rAAV) expressing ASPA can cure Canavan disease. Mice with CD administered rAAV expressing ASPA by intraventricular injection show similar improvement in motor function (FIG. 8). Systemic (e.g., IV) injection of rAAV-ASPA also expands the treatment window. Experimental data indicate that rAAV-ASPA administered intravenously to Nur7 mice (a model of mild CD) beginning at 3 months of age results in restoration of mobility (FIG. 9) and improvement in cognitive function (FIG. 10). Furthermore, intravenous administration of rAAV-ASPA results in rapid and efficient clearance of CNS spongy degeneration (FIG. 11) in Nur7 mice receiving treatment at P1. Importantly, intravenous administration of rAAV-ASPA results in a rapid reduction of NAA in the brain of Nur7 mice (Fig. 12). Mice were treated at 6 weeks and monitored at 7 and 10 weeks by neuroimaging. Treatment of CD mice with rAAV-ASPA also restores the lipid profile of myelin, as demonstrated by measurement of sphingolipids in treated and control mice (Fig. 13).

A crucial finding is that high ubiquitous expression of ASPA improves motor performance in treated CD mice compared to wild-type mice. This may be the result of improved energy metabolism due to direct intervention in ASPA-mediated NAA metabolism.

The results of this global metabolomic study compare brain samples from WT or aspartoacylase (ASPA) KO, or recombinant AAV-treated KO mice (to express ASPA or not, as control), including changes in metabolites related to energetics (carbohydrate and lipid metabolism), neurotransmitter production, inflammation, and redox homeostasis. In principal component analysis (PCA), samples were divided into two groups, with WT and rAAV Tx in one, and KO and rAAV Ctrl in the other, indicating that rAAV Tx “rescued” the metabolomic effects of ASPA deficiency. Consistent with the loss of ASPA function, N-acetylaspartate (NAA) accumulated in the brain (KO vs WT), whereas levels decreased upon ASPA re-expression (rAAV Tx vs Ctrl). Lipids tended to show decreases across all classes, potentially reflecting changes in beta-oxidation and/or biosynthesis (KO vs WT); rAAV Tx (compared to rAAV Ctrl) showed increases in a marker of lipid biosynthesis, with increases in several lipid classes. Evidence for decreased glycolytic usage in KO (compared to WT) was reversed in rAAV Tx (compared to rAAV Control). Finally, changes in the dataset pointed to increased inflammation and oxidative stress in KO (compared to WT), with decreases in rAAV Tx (compared to Ctrl).

Summary of results

Regarding gene therapy:

Gene therapy reverses metabolic changes in Canavan disease brains.

Regarding the pathomechanism of Canavan disease and CNS energy metabolism:

1. Glucose metabolism in the brain with Canavan

a. Substrates for glycolysis are abundant in Canavan mouse brains i. Substrates for glycolysis accumulate (e.g. glucose, glucose-6-phosphate, 3-phosphoglycerate, phosphoenolpyruvate), indicating a decreased rate of glycolysis. ii. Increased phosphoenolpyruvate inhibits the enzyme “triose phosphate isomerase”, decreasing the efficiency of glycolysis by utilizing only 50% of each glucose molecule for energy production.

iii. Glycogen, the glucose storage system of cells, is being broken down despite the abundance of glucose in the brains of Canavan mice. In a physiological state, increased glucose leads to glycogen synthesis rather than its breakdown.

b. Glycolysis products are unchanged or decreased in Canavan i-treated mouse brains. Pyruvate is unchanged despite the abundance of glycolysis substrates, which is paralleled by a decrease in lactate. Both indicate that the glycolytic rate is decreased.

2. Fatty acid metabolism in the brain with Canavan

a. The products of fatty acid breakdown are modified to promote beta-oxidation (use of fatty acids for energy production).

i. Carnitine is increased in Canavan brains. Transport to mitochondria is the rate-limiting step in beta-oxidation. This transport is performed by carnitine palmitoyltransferase 1 and 2 (CPT1 and CPT2). Fatty acids need to be bound to carnitine in order to be transported into mitochondria. An increase in carnitine usually facilitates esterification of fatty acids and carnitine-supported transport of fatty acids into mitochondria.

ii. Several carnitine esters are increased, decreased, or unchanged, indicating fatty acid consumption in the brains with Canavan.

iii. Several fatty acids are also decreased in Canavan brains, which could be due to the consumption of fatty acids for energy production, e.g. ATP or other energy equivalents.

iv. Malonylcarnitine, a surrogate marker for malonyl-CoA, is decreased, facilitating fatty acid transport into mitochondria for beta-oxidation (malonyl-CoA inhibits CPT1 and thus reduces fatty acid transport into mitochondria; a reduction in malonyl-CoA removes this inhibitory stimulus). Malonyl-CoA is also a precursor for fatty acid synthesis and thus mediates between fatty acid degradation and fatty acid synthesis. The fact that malonylcarnitine is decreased points to fatty acid degradation.

3. Ketone bodies in brains with Canavan

a. Increases the ketone body beta-hydroxybutyrate, which breaks down directly into acetate and enters the TCA cycle.

b. Beta-hydroxybutyrate also mediates between metabolism and transcription.

4. Acetate in brains with Canavan

a. Acetyl-CoA is not modified in the neurometabolome of CD, arguing against the “acetate deficiency hypothesis.” Furthermore, it might also explain why acetate supplementation failed to cure Canavan disease in patients.

b. Acetylcarnitine, which has been reported to be crucial in energy production, is greatly increased and therefore facilitates energy production.

5. Antioxidants

a. Some antioxidants decreased, others increased, which could support the oxidative stress hypothesis. However, there was a significant reduction in metabolites for antioxidant synthesis, indicating that the decrease in some antioxidants is a matter of supply rather than demand. This argues against the oxidative stress hypothesis as the main causative factor of the disease.

NAA accumulation and/or ASPA deficiency alter CNS energy metabolism by favoring fatty acids over glucose/lactate for energy production, leading to "self-consumption" of fatty acids, critical components of myelin, and thus white matter vacuolations and disease pathology. Metabolic deficiency of NAA and/or its causal ASPA deficiency could promote consumption of fatty acids instead of glucose/lactate for energy production.

NAA metabolism with its associated proteins such as AspA could be a key player in regulating and communicating between metabolic pathways and in monitoring metabolic homeostasis of cells and organs, as demonstrated by the fact that despite glucose abundance, fatty acid metabolism is favored, glycogen is broken down, and ketone bodies are formed, which are highly detrimental processes in a physiological system, but not in an altered NAA/ASPA metabolism state. Furthermore, besides being involved in NAA metabolism, AspA may play critical roles in energy metabolism. These conclusions are reinforced by the fact that rAAV-mediated ASPA administration corrects these observed changes.

Example 2: Nur7 mouse model of Canavan disease

A single intravenous (i.v.) injection of recombinant adeno-associated virus (rAAV) expressing human ASPA (hASPA) rescues early lethality and partially restores motor function (1st generation gene therapy) in a CD knockout (CD KO) mouse, which resembles the congenital subform of CD and displays the most severe phenotype of all available CD mouse models, with early death around postnatal day (p) 28. This example describes a 3rd generation rAAV expressing hASPA (also termed FKzhAspA-Opt), comprising the sequence represented by SEQ ID NO: 1, and cures the disease in a CD KO mouse model. Interestingly, the 3rd generation gene therapy converts CD KO mice into “super mice”, which outperform wild-type (WT) mice in the rotating cylinder motor function test. This rescue is persistent: treated mice evaluated at 1.5 years of age continue to show no signs of disease recurrence. CNS pathology and magnetic resonance imaging (MRI) at p25 and p365 show complete normalization.

To further support the efficacy of the 3rd generation gene therapy, neurometabolomic profiling was performed. The data indicate that over 400 characterized metabolites that showed a reversal of Canavan disease-related metabolic changes, including myelin-associated lipids. Transcriptomic profiling was also performed.

To further evaluate the potency of the 3rd generation gene therapy, different doses and routes of administration were tested. Notably, rAAV administered intraventricularly (ICV) at 200-fold lower doses still rescued lethality, while mice treated ICV at 20-fold lower doses matched WT mice in motor function tests.

Next, the Nur7 mouse model, which resembles the infantile and juvenile subform of CD, was tested. This model shows a similar disease pattern to the CD KO mouse with respect to growth curve and neurological symptoms, but eventually regains weight and shows survival similar to wild-type mice (FIG. 14). Again, mice received a single i.v. dose of rAAV-hASPA at p1 (positive reference control) and subsequent groups were dosed at 6, 12, and 24 weeks of age with a dose 10-fold higher than that of neonates to determine the therapeutic window. Notably, mice treated at 6 weeks of age recovered within 4 weeks of treatment. Mice treated after 6 weeks take longer to recover but still showed significant improvement relative to Nur7 mutants (FIG. 15). This recovery also correlated with CNS pathology and MRI.

Motor function was assessed in all mice 4 weeks after treatment and at subsequent intervals through 1 year of age for direct comparison (FIG. 16 and FIG. 17). In general, the earlier mice were treated, the better the therapeutic outcome. Remarkably, 6-week-old juvenile mice fully recovered within 4 weeks of injection (FIG. 16 and FIG. 17). Although mice treated at 3 months of age and thereafter did not respond immediately in the first 4 weeks after treatment, they ultimately showed significant improvements relative to Nur7 mutant control mice. Cognitive function was also assessed; representative data are shown in FIG. 18. Notably, cognitive function testing revealed that treated mice recover cognitively before motor function improves; this was true even at late time points after treatment. Furthermore, response to rAAV-hASPA gene therapy was confirmed by MRS for N-acetylaspartate, MRI, and neuropathology (FIG. 19-21).

Overall, the data demonstrate that rAAV-mediated hASPA expression from the 3rd generation gene therapy vector not only prevents but also rescues the clinical manifestation and pathology of the juvenile and adult model of Canavan disease at an unprecedented level, which could have implications for other CNS disorders requiring treatment in later life stages. Furthermore, this is confirmed at different levels of cellular complexity by MRI, fMRI, CNS pathology, and neurometabolic profiling.

Example 3: Expression of hASPA restricted to astrocytes in CD KO mice (severe phenotype)

Several tissue/cell-specific expression cassettes configured to restrict hASPA expression to astrocytes, neurons, oligodendrocytes, liver, heart, or muscle were produced. For example, a rAAV-hASPA construct comprising an astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was produced. Tissue-restricted rAAVs were administered to Canavan disease knockout (CD KO) mice. Strikingly, mice expressing peripheral organ-restricted hASPA showed increased survival and normalization of the growth curve at later time points, indicating a contribution of peripheral organs to the disease pathomechanism.

Astrocyte-restricted hASPA expression produced the greatest disease recovery, matching the performance of wild-type (WT) mice (FIG. 22-24). FIG. 22 shows the results of astrocyte-restricted hASPA expression on the survival and growth of treated CD KO mice compared to untreated control mice. FIG. 23 shows that astrocyte-restricted hASPA expression results in restoration of motor function in treated CD KO mice as measured by the rolling cylinder test at p27 and p90. FIG. 24 shows that astrocyte-restricted hASPA expression results in restoration of motor function in treated CD KO mice as measured by the balance beam test at p27 and p90.

Mice were administered a lower dose of rAAV-hASPA via localized brain injections. The data indicate that the elimination of the localized T2 hyperintensity signal on MRI correlated well with the reduction of NAA levels by MRE (FIG. 25). In other words, the farther away from the injection site, the higher the NAA levels were, supporting the idea of drainage and hydrolytic activity of NAA towards the injection side. We are currently investigating this metabolic sink theory in more detail by creating a functional map of therapeutic gene transfer in the brain using mass spectrometry quantification of NAA, and analyzing the vector genome and ASPA transcripts in different anatomical regions.

Overall, the data indicate that restoration of hASPA expression in oligodendrocytes is not necessary to rescue lethality and the Canavan disease phenotype.

Example 4: High-field in vivo neuroimaging

Gene therapy targeting the central nervous system (CNS) is one of the most challenging gene therapies due to the blood-brain barrier (BBB). One obstacle in monitoring and evaluating CNS gene therapy is the noninvasive assessment of therapeutic outcome. Although CNS biopsies and sections are the gold standard for assessing brain pathology and response to CNS gene therapy, their invasive nature and potentially associated complications limit their frequent use in preclinical and clinical studies.

This example describes high-field in vivo neuroimaging to monitor rAAV-based CNS-targeted gene therapy administered intravenously (i.v.) and intracerebroventricularly (i.c.v.) in a mouse model of Canavan disease (CD). Characteristically, Canavan disease presents with a very high peak of NAA detected by magnetic resonance spectrometry (MRS) and hyperintensity on T2-weighted anatomical images using magnetic resonance imaging (MRI). Accordingly, the efficacy of i.v. and i.c.v. gene therapy by these two means was evaluated. Consistent with the data on motor function and pathology described elsewhere in the disclosure, both MRI and MRS alterations were completely normalized by gene therapy.

Another characteristic neuropathological change in Canavan brain sections is the loss of white matter bundles, which is thought to explain the neurological symptoms observed in Canavan disease patients. The ability of diffusion tensor MRI (DTI) to allow assessment of white matter bundle degeneration and recovery following gene therapy without brain biopsies was investigated. Selecting the thalamus and corpus callosum as regions of interest (ROI), DTI shows a recovery of cerebral white matter integrity when using the 3rd generation Canavan gene therapy (e.g., FKzhAspA-Opt, SEQ ID NO: 1). Furthermore, the 3rd generation gene therapy converts this CD mouse model with the most severe phenotype into a “super mouse,” outperforming the wild-type mouse in motor function tests.

Functional connectivity identifies brain regions that not only show response to treatment, but also indicate possible explanations for this enhanced phenotype. Using resting-state functional MRI (RS-fMRI), treated CD mice were shown to display a pattern of functional connectivity more similar to, or even superior to, that observed in the WT brain. This indicates that facilitated functional connectivity between brain regions could provide a neural mechanism underlying the observed enhanced motor function.

In summary, the imaging data show that high-field in vivo neuroimaging is a valuable tool for detailed monitoring of preclinical gene therapy and CNS pathology, can provide insight into pathophysiology, and has potential implications for use in predicting and evaluating clinical trial outcomes.

Example 5: Reorientation of N-acetylaspartate metabolism in the central nervous system normalizes myelination and cures Canavan disease.

Materials and methods

Procedures with animals

Heterozygous Aspa/− mice were bred on a Sv129 background and newborns were genotyped on the day of birth. Briefly, 1 mm tail tips were clipped and genomic DNA was extracted according to the manufacturer’s protocol using the manual QIAamp DNA minikit or the QIAcube robot (Qiagen, Hilden, Germany). DNA extraction was followed by quantitative PCR (qPCR). Injections were made in P1 via the right facial vein with a genome copy number (GC) of 4*10A11 (~2.6*10A14 vg/kg; based on an average weight of 1.5 g), 1.33*10A11 (~8.8*10A13 vg/kg; based on an average weight of 1.5 g), or 4x10A10 (~2.6*10A13 vg/kg; based on an average weight of 1.5 g). After each procedure, the pups were cleaned with 70% ethanol and rubbed with bedding material. The parent animal was returned after a brief numbing of the nose with 70% ethanol. Vg = viral genomes

Viral production and vector design

Recombinant adeno-associated virus (rAAV) was produced by transient transfection of HEK 293 cells and cesium chloride (CsCl) sedimentation. Vector preparations were titered by quantitative PCR, and purity was assessed by 4–12% SDS-acrylamide gel electrophoresis and silver staining (Invitrogen, Carlsbad, CA). Morphological integrity of virions was assessed by negative-stain rAAV transmission electron microscopy. Due to size restrictions of encapsidation, a single-stranded rAAV genome was used for the phGFAP-hASPA and phGFAP-EGFP constructs. All other vectors were self-complementary (sc) AAV vectors (scAAV).

Western Transfer

Proteins were extracted using RIPA buffer. Protein quantification was performed using the BCA assay (Pierce Biotechnologies, Rockford, IL, USA) and 10–20 pg of total protein mixed with 4X Laemmli buffer (BioRad, Hercules, CA, USA) was loaded onto a 10–12% Tris-HCl acrylamide gel (BioRad, Hercules, CA, USA). After electrophoresis, proteins were blotted onto a nitrocellulose membrane (BioRad, Hercules, CA, USA) using the Trans-Blot Turbo Transfer System (BioRad, Hercules, CA, USA). Membranes were then blocked at room temperature for at least one hour with Odyssey blocking buffer (Licor, Lincoln, NE, USA). The membranes were then incubated with the primary antibody (anti ASPA, 1:2000, ab 97454; anti-actin, 1:5000, ab8224) at 4 °C overnight and incubated with the secondary antibody (Licor, Lincoln, NE, USA) the next day. The membranes were analyzed with the Odyssey analyzer (Licor, Lincoln, NE, USA). Quantification was performed using ImageJ.

Isolation of brain regions and extraction of DNA and RNA

Mice were anesthetized with isoflurane and transcardially perfused with ice-cold phosphate-buffered saline (PBS). The brains were then removed and split in half along the interhemispheric fissure. One brain half at a time was placed on an ice-cold, RNase-free dish under a dissecting microscope. First, the olfactory bulb was removed with a cold razor blade. Next, the brain stem/midbrain was removed along the line between the cortex/thalamus and the quadrigeminal lamina. The brain stem was subdivided into the midbrain, quadrigeminal lamina, cerebellum, and brain stem. In addition, the thalamus/hypothalamus was removed with a Wecker dissection microspatula (Roboz Surgical Instruments Inc., Gaithersburg, MD, USA). The hippocampus was removed. Finally, part of the cortex was removed with a new razor blade. All samples were frozen immediately after removal.

DNA and RNA were extracted using the Qiagen Allprep DNA/RNA Mini kit (Qiagen, Hilden, Germany) and RNA samples were subjected to on-column DNase treatment prior to RT-PCR. DNA was subjected to viral genome copy number determination and total RNA to RT-PCR (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems).

Droplet digital PCR (ddPCR)

Multiplex ddPCR was performed on a QX200 ddPCR system (Bio-Rad, Hercules, CA). All assays were based on TaqMan probes, where the probes of the gene of interest were labeled with FAM and the reference gene as VIC. Bio-Rad ddPCR mastermix without dUTP (BioRad 1863024) was used for all ddPCR reactions.

Number of copies of the vector genome

DNA was digested with BamHI at >10 U/pg DNA at 37 °C for 1 h. BamHI digestion ensured single copies of the rAAV genomes. All vectors contained an RBG sequence, which was selected for viral genome quantification. The number of viral genomes was normalized to the number of diploid cells using the transferrin receptor (Tfrc) as a reference gene (Invitrogen, 4458367).

Motor function and spatial memory tests

Motor performance of mice was assessed using accelerated rotating cylinder for motor function and endurance, balance beam for vestibular function and ataxia, and inverted screen for grip strength. For each motor function test, n=8 mice were injected and tested independently.

Accelerated rotating cylinder

Mice were trained two days before the test day for three sets each. On the test day, mice were placed in the rotating cylinder for acclimatization for 1 minute. Each mouse was tested three times and the best value was used for analysis. Acceleration and time were set between 4 and 40 rpm for 5 minutes.

Balance beam

To increase the rigor of this test, the time limit was increased from 3 to 5 minutes. Mice were placed in the center of the balance beam and the latency to landing was measured. Again, the best value was counted.

Inverted screen

Mice were placed in the centre of a grid (30 cm2 with 25 mm2 holes) in a horizontal position and allowed to acclimatise for 1 min. The grid was slowly rotated over 15 s to 125 degrees so that the mouse was hanging upside down. The time to fall was measured. The time limit for the p28 test was 3 min. At all other time points the time limit was 5 min to increase the rigour of the test.

T-maze

The T-maze test was performed spontaneously and without reward, with all arms of the T-maze open during the test. Mice were placed in the start chamber with the door down and the side arm doors open for 10 s, after which the start chamber door was opened and they were allowed to enter and explore the T-maze. When the mouse fully entered one of the side arms, defined as when all four of its paws passed over the edge of the arm, all T-maze doors were closed and the mouse was returned to the start chamber for a 10 s rest time. During these 10 s, the side arm doors were opened again. This process was repeated 10 times, giving the mouse a total of 10 opportunities to alternate its side arm choice. The final result is expressed as a quotient of the number of alternations over 10.

H&E staining and Luxol fast blue

Mice were sacrificed and transcardially perfused with ice-cold PBS and 4% paraformaldehyde (PFA). Tissues were removed and sliced using an Alto Brain/Spinal Cord Matrix (Roboz Surgical Instruments Inc., Gaithersburg, MD, USA). Mouse tissues were subsequently stored in PFA at 4 °C overnight. Paraffin embedding, hematoxylin and eosin (H&E) staining, and Luxol fast blue staining were performed. Stained sections were analyzed and imaged with an Axioscope 50 (Zeiss, Jena, Germany) using a DMC2900 camera (Leica Microsystems, Wetzlar, Germany).

Magnetic resonance imaging (MRI) and spectroscopy (ERM)

Mice were anesthetized with 2% isoflurane and vital signs were continuously monitored throughout imaging. Mice were imaged at P42 with a 4.7T/40 cm horizontal magnet (Oxford, UK) equipped with a Bruker Biospec Avance console (Bruker, Germany). Experiments for all other images were performed using a 4.7T/40 cm horizontal magnet (Oxford, UK) equipped with a Bruker Biospec Avance III HD console (Bruker, Germany). A 1 H radiofrequency mouse head coil (Bruker, Germany) with an inner diameter of 23 mm was used for experiments.

T1-weighted anatomical images were acquired using the FLASH sequence with the following parameters: repetition time (TR) = 280.86 ms, echo time (TE) = 4.5 ms, matrix size = 384x384, field of view (FOV) = 18x18 mm2, number of slices = 15, slice thickness = 0.5 mm, flip angle = 40°, number of averages = 8. T2-weighted images were acquired using the TurboRARE sequence with TR = 2200 ms, TE = 36 ms, echo spacing = 12 ms, 8 averages, and rarity factor = 8. 1H magnetic resonance spectroscopy data were acquired using single-voxel PRESS (speckle-resolved spectroscopy) sequence (repetition time = 2500 ms, echo time = 16 ms, number of averages = 8). Functional MRI images were acquired over 10 minutes using echo-planar imaging (EPI) sequence, with TR=1000 ms, TE=18 ms, matrix size=96x96, FOV=18x18 mm2, number of slices=15, slice thickness=0.5 mm, number of repetitions=600. Diffusion tensor MRI (DTI) data were acquired from 30 directions with B value of 650/0, TR=2300 ms, TE=21 ms, number of averages=4, with the same geometry parameters as EPI.

1H magnetic resonance imaging and spectroscopy study.

Proton spectra were fit using LCModel (Version 6.2-2B) which analyzed in vivo proton spectra as a linear combination of in vitro model spectra from individual metabolite solutions (Provencher, 2001) and generated data as absolute fits (in institutional units) and % SD. SD was used as a measure of fit reliability. Spectral inclusion criteria were SD <20% for NAA, creatine, and inositol.

Resting state functional connectivity (CFer) analysis

EPI images were preprocessed using Medical Image Visualization and Analysis (MIVA, http://ccni.wpi.edu/) and Matlab 2010b (the Mathworks Inc.). All EPI images were first registered to a standard anatomy, where seed regions were defined. Following registration, all EPIs underwent motion correction, spatial smoothing (full-width-half-maximum = 1 mm), and 0.002–0.1 Hz bandpass filtering. Seed-based CFer was calculated using a previously demonstrated algorithm.

Diffusion tensor magnetic resonance imaging (DTI)

DTI data were analyzed using DTIstudio (www.mristudio.org/, Susumi Mori and Hangyi Jiang, Johns Hopkins University), including eddy current correction, motion correction, and generation of all tensor metrics (FA and voxel-wise diffusion tensor eigenvalue decomposition). FA values in selected regions of interest (ROIs) were extracted from manually drawn ROIs.

For all imaging outcomes, group comparisons were performed using one-way ANOVA, with a significance threshold of p<0.05.

Immunohistology

Mice were transcardially perfused with 4% paraformaldehyde (PFA) and maintained in PFA overnight at 4°C. The next day, brains were removed and subjected to sucrose gradient steps (10, 20, and 30%) overnight at 4°C. Brains were mounted in O.T.C. compound (Fisher HealthCare, Houston, TX, USA) and stored at -80°C until cryosectioning (Cryostar NX70, Thermo Fisher Scientific, Walldorf, Germany). Floating brain slices were washed in 1X PBS 3X for 5 minutes each. Cells were permeabilized with 1XPBS and 0.5% Triton-X 100 at room temperature for 1 h, followed by blocking for 1 h at room temperature with 5% serum (10% normal goat serum, Life Technologies, 50062Z). Brain sections were incubated with primary antibodies (anti-GFAP, EMD Millipore, 1:1000, MAB360; anti-MBP, Abeam, 1:1000, ab40390) in 1.5% serum overnight at 4 °C, washed the next day (1xPBS, x3, 5 min each), and stained with secondary antibody in 1.5% serum at room temperature for 1 h (anti-mouse or rabbit; Invitrogen, A-11031 or A-11011). Sections were mounted with Vectashield with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA).

Brain sections were viewed and recorded with the DM 5500B Upright microscope (Leica Microsystems, Wetzlar, Germany) and the Leica DFC365 FX digital camera.

Computer programs and statistics

Image analysis and visualization were performed using Imaris 8.2 software (Bitplane Inc., South Windsor, CT, USA). Western blots were quantified using ImageJ (National Institute of Health, USA). Graphs were analyzed and statistical calculations were performed using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). The correlation between global functional connectivity on fMRI and mean accelerated spinning cylinder performance was calculated and linear regression analysis was performed using GraphPad Prism 7. Statistics were performed using GraphPad Prism 7, two-way ANOVA with multiple comparisons correction (Turkey) for weights, and one-way ANOVA with multiple comparisons correction for all other statistics, if not otherwise stated. If not otherwise stated, at least n=3 mice were analyzed.

Optimization of the transgenic cassette achieves a rapid therapeutic response in Canavan mice

To overcome the challenge of efficacy and sustainability of a 1st generation gene therapy, hASPA expression was increased from the vector genome without changing the parameters of the administration route, vector dose, or serotype. Comparing the effect of Kozak sequence and cDNA optimization, two new expression cassettes with a half- or full-length Kozak sequence and codon-optimized cDNA were designed, which were termed 2nd and 3rd generation vectors (FIG. 26A; hereafter referred to as 2nd or 3rd generation), respectively. To test in vivo translatability, each of the expression cassettes was encapsidated into the highly CNS-atopic rAAV9 vector for intravenous gene delivery to the CNS. Untreated and 1st generation-treated mice showed the characteristic low weight around p14–16 (FIG. 27). However, mice treated with the 2nd and 3rd generations (n=10 each) showed weight gain parallel to that of wild-type (WT) mice, indicating a more rapid onset of therapeutic transgene expression levels (FIG. 27). To assess neuropathology in live mice, MRI was performed at p42 for treated animals and at p28 for untreated animals. While untreated and 1st generation mice showed strong hyperintense signals (white signals) on T2-weighted sequences, particularly in deeper brain regions, signals appeared isotense (unremarkable) in the 2nd and 3rd generation vector-treated groups (FIG. 28), indicating normalization of brain edema. This therapeutic effect was corroborated by the normalization of NAA levels in the 2nd and 3rd generation treated groups in the ERM, which also coincided with ASPA protein expression levels (FIG. 26B-26C). Finally, the neurohistopathology of the mice was assessed at p25. Again, 1st generation treated mice still showed CNS vacuolization, although to a lesser extent than untreated mice. In contrast, the brains of 2nd and 3rd generation treated mice were indistinguishable from WT, demonstrating that the enhanced gene therapy effectively mitigates the neuropathology (FIG.

29).

3rd generation of Canavan-treated mice sustainably outperform control animals

CD KO mice recapitulate the clinical phenotype of Canavan patients, exhibiting ataxia, imbalance, muscle weakness, and cognitive impairment within the first month of life. At 1 month of age, 2nd generation-treated CD mice were found to perform as well as WT controls on the accelerated rotating cylinder (FIG. 30A). The 3rd generation-treated group displayed a “supermouse” phenotype, significantly outperforming WT mice throughout the year-long duration of the study (FIG. 30A and FIG. 31). To assess ataxia and balance ability, 2nd and 3rd generation-treated mice were tested on the balance beam, also showing complete recovery (FIG. 30B and FIG. 31). This paralleled performance on the inverted screen and was persistent throughout the study (FIG. 30C and FIG. 31). Finally, to determine the long-term ability to retrieve spatial/working memory, mice were tested in the T-maze at 1 year, performing as well as WT control mice (FIG. 32).

Efficient delivery of the hASPA gene to the CNS persistently eliminates neuropathology and normalizes NAA levels

To determine whether phenotypic rescue of psychomotor function was supported by brain pathology and NAA biomarker levels, live mice from all three treatment groups were assessed at 1 year of age. First, T2 MRI showed strong hyperintensities in 1st generation-treated mice, particularly in the thalamus, midbrain, and cerebellum (Fig. 33A). In contrast, 2nd generation-treated mice showed mildly increased T2 signals in the midbrain, being otherwise similar to WT mice. Importantly, comparison between WT and 3rd generation-treated mice revealed no differences in T2 MRI, which was also corroborated by neuropathological analysis (Fig. 33B). Although some CNS regions (e.g., thoracic spinal cord) showed similar patterns between WT and all three treatment groups, brain regions with the most intense T2 signal also corresponded to the most severe vacuolization in brain sections from 1st generation-treated mice (FIG. 33A-33B and FIG. 34). When 1-year-old mice from all treatment groups were subjected to MRS for NAA quantification, 1st generation-treated mice showed significantly higher NAA signal than WT mice, whereas NAA levels from 2nd and 3rd generation-treated mice normalized (FIG. 33C). This strongly indicates that new generation gene therapies are significantly more effective and are able to normalize NAA biomarker levels, a finding that is further supported by normalized urinary NAA levels, as measured by mass spectrometry (FIG. 33D).

Astrocyte-specific ASPA expression is sufficient to generate the “supermouse phenotype”

We investigated how 3rd generation gene therapy was able to improve performance beyond that of WT control animals. No phenotypic differences were observed between patients heterozygous or homozygous for the WT ASPA allele, implying that oligodendroglial ASPA and its associated NAA catabolism might not cause dose-dependent behavioral variations within the physiological range. Furthermore, most rAAVs, including rAAV9, poorly transduce oligodendrocytes. Thus, in some embodiments, expression of the hASPA transgene from non-oligodendrocyte glial cells contributes to the “super mouse” phenomenon observed in the accelerated rotating cylinder. To test whether WT mice respond to ASPA supplementation with increased motor performance, WT animals were treated with 3rd generation gene therapy. Initially, treated WT mice showed no differences in accelerated rolling cylinder at p28, but began to significantly outperform untreated WT controls between p90 and 1 year of age, indicating that supplementation of non-ASPA-expressing cells with ASPA via gene transfer contributes to the observed "enhanced" phenotype (FIG. 35).

To further define the CNS cell type contributing to the “supermouse” phenotype, the 3rd generation hASPA construct was paired with a partial human glial fibrillary acidic protein (phGFAP) promoter. First, the astrocyte specificity of the phGFAP promoter was confirmed by expressing enhanced green fluorescent protein (EGFP), which showed colocalization with glial fibrillary acidic protein (GFAP)-positive cells, but not with myelin basic protein (MBP)-positive cells (FIG. 36A-36B). Next, neonatal CD KO mice were treated with rAAVphGFAP-hASPA-Opt. Treated mice showed the same growth curve as the ubiquitously expressed 3rd generation gene therapy-treated group (FIG. 36C). The performance of these mice on the accelerated rotating cylinder was significantly increased relative to WT mice at all testing time points (e.g., p27 and p90), similar to what was observed with the 3rd generation vector for ubiquitous expression of hASPA (FIG. 36D). These data indicate that astrocyte-restricted hASPA expression contributes substantially to improved performance on the accelerated rotating cylinder. Other less stringent motor function tests were observed to show no differences compared to WT mice (FIG.

36D). Importantly, rAA VphGFAP-hASPA-Opt-treated mice performed better than untreated control mice in the T-maze but showed no differences in spatial/working memory function versus WT animals (FIG. 36E and FIG. 32). This indicates that mice with increased motor performance have normal spatial/working memory function in the T-maze test. Furthermore, MRI and MRS showed normalization of T2 signals and NAA levels in the brains of mice receiving astrocyte-specific gene therapy (FIG. 36E-36F), indicating that astrocyte-restricted hASPA expression alone is capable of creating an alternative metabolic sink for NAA and is rescuing neuropathology and biomarker expression in CD mice. To corroborate that astrocyte-restricted hASPA expression can rescue neuropathology, e.g., improve myelination, brain sections were stained with Luxol fast blue for myelin sheaths. The staining pattern was indistinguishable between WT, astrocyte-restricted, and 3rd generation gene therapy-treated animals with ubiquitous expression, and all restored myelination compared to untreated CD KO mice (FIG. 36I).

Optimized gene therapy effectively rescues Canavan phenotype at lower doses

An important aspect in the translation of gene therapy to the clinic is the vector dose, which is relevant for manufacturing burden, costs, and safety. Based on the performance of mice treated with a full dose of the hASPA construct, e.g., 4X1011 genome copies (CG)/animal, 3-fold (1.33x10” CG) and 10-fold (4x1010 CG) lower 3rd generation gene therapy doses were tested to compare their therapeutic outcomes. Within the first 4 weeks of life, mice treated with 3- and 10-fold lower 3rd generation doses showed significantly better weight gain than mice treated with full and 3-fold lower 1st generation doses (FIG. 27 and FIG. 37). This was further demonstrated over the course of the entire study period for the 3-fold lower 3rd generation dose group, which paralleled the weights of WT animals. In contrast, mice treated with a 3-fold lower dose of 1st generation decreased starting at 16 weeks of age (FIG. 37). Again, mice in the study were subjected to MRI and ERM for in vivo assessment of the CNS, but at an earlier time point (p25). T2 sequences showed hyperintensities particularly in the thalamus, midbrain, cerebellum, and brainstem of untreated mice (FIG. 38A). This signal was reduced in a dose-dependent manner, with the 3-fold lower dose group showing the least hyperintense signal. As a control, WT and CD mice treated with full dose showed normalized T2 signals and were indistinguishable from each other (FIG. 38A). The MRI findings from the dose-reduction study were also reflected in the quantification of NAA by ERM (FIG. 38B). This was further supported by neuropathology analysis, showing a dose-dependent distribution of vacuoles with a similar pattern in 1st generation full-dose treated mice and 3rd generation 10-fold lower-dose treated mice (FIG. 38C and FIG. 39). Lastly, mice were subjected to accelerated rotating cylinder, balance beam, and inverted screen tests. At p27, 3rd generation 10-fold lower-dose treated mice performed as well as WT mice on the rotating cylinder and balance beam, but not as well on the inverted screen test (Fig. 38D). Over time, this low-dose treated group showed impaired motor function at p90. In contrast, at p27, a 3-fold lower dose could restore the motor functions of CD KO mice to the levels of WT mice, as measured by all three aforementioned tests. At p90, these mice continued to perform as well as WT mice on the accelerated rotating cylinder and balance beam, but not on the inverted screen (FIG. 38D). These data indicate that the 3rd generation gene therapy is significantly more potent, even at lower doses, than our 1st generation gene therapy.

CNS region-specific rAAV genome distribution profile is consistent with regional neuropathology

Since expression cassette optimization was found to correlate with increased hASPA protein expression (FIG. 26B), 11 CNS regions were analyzed to gain further insight into CNS region-specific pathology and rAAV transduction. No significant differences in the number of vector CGs per diploid cell were detected between the 1st and 3rd generation full-dose treatment groups, indicating that increased hASPA protein expression from the optimized expression cassette, but not improved vector genome delivery, was responsible for the significant therapeutic improvement of 3rd generation-treated mice (FIG. 26B and FIG. 40). When regional distribution of the vector genome was categorized, the cortex showed the highest number of rAAV CGs per cell and the cerebellum the lowest (FIG. 40). In relation to neuropathology, this indicates that certain brain regions have different therapeutic thresholds to achieve complete mitigation (FIG. 33A-33D and FIG. 34). In particular, the cerebellum showed minimal response when comparing 1st generation full dose gene therapy to 3rd generation 10- and 3-fold lower dose gene therapy (FIG.

39). However, with full-dose 3rd generation treatment, complete rescue was achieved with lower rAAV GC numbers than in any other CNS region (FIG. 39 and FIG. 40).

High-field neuroimaging enables noninvasive monitoring and prediction of therapeutic outcomes

One aspect of CD is the loss of myelin structures in the CNS. Myelin staining with Luxol fast blue showed widespread vacuoles and reduced myelin in untreated CD KO mice (Fig. 41). In contrast, myelin structures in WT mice and mice treated with full doses of the 3rd generation were indistinguishable (Fig. 41). To assess whether these findings can be monitored in the living mouse, high-field DTI was applied to assess myelin fiber bundles in the corpus callosum (CC) and external capsule (EC). Tractography to obtain a global assessment of fiber bundle morphology showed substantially altered and shortened interhemispheric bundles of the CC in untreated mice, whereas WT and 3rd generation-treated mice were indistinguishable (Fig. 42A). For quantitative assessment, fractional anisotropy (FA) values of the right and left CC and CE were compared between groups. While there were no significant differences between WT and treated mice, untreated mice differed significantly, supporting the tractography data and indicating the value of DTI for noninvasive assessment of CNS gene therapy (FIG. 42B).

Resting-state functional MRI (ER-fMRI) was performed on groups of WT, untreated, and treated male mice to determine whether ubiquitous ASPA expression changes functional connectivity. A total of 19 different brain regions were analyzed (FIG. 43). Based on T-score analysis, the most active brain regions were seen in untreated mice, with decreased activity in WT and treated mice, indicating that the ASPA-deficient brain may have to engage more brain regions at baseline (FIG. 42C-42D). Thus, in some embodiments, ubiquitous ASPA expression in the brain facilitates communication between brain regions and therefore engages fewer brain regions at baseline (FIG. 42C-42D). Correlation analysis between the mean number of active brain regions and the accelerated rotating cylinder results revealed a negative correlation between global functional connectivity and accelerated rotating cylinder motor function, indicating that ER-fMRI supports the correlation between treatment and motor performance (R2 = 0.89; FIG. 42D-42F).

Example 6: NAA levels correlate with T2 signal intensities

Mice were treated IV at juvenile age and monitored for four weeks by MRI/MRA. Brain NAA levels and corresponding T2-weighted MRI sequences indicate that the hyperintense signal decreases when NAA levels decrease in mice treated at 6 weeks of age (FIG. 44). This indicates that neuroradiological correlates of pathology can be reversed when treated after the onset of pathology.

Example 7: Myelin restoration after treatment with hASPA gene therapy

Six-week-old ASPA-deficient mice (juvenile) display altered axon and myelin structure (FIG. 45, left). Four weeks after treatment, treated mice display axonal patterning and myelination indistinguishable from wild-type mice, indicating reversal of neuropathology (FIG. 45, right).

Mice were treated at 6 weeks of age and sacrificed at 7 or 10 weeks for analysis of the anterior commissure by electron microscopy. The G ratio describes the relationship between the inner and outer diameter of the axon and is indicative of the thickness of the myelin and the axon. The lower the value, the greater the presence of myelin. The data indicate that at 7 weeks of age untreated mice (Nur7) have a significantly higher g ratio than wild-type mice; this is also observed in mice one week after treatment (FIG.

54). After 4 weeks of treatment (10 weeks of age), treated mice show a significantly lower g ratio than untreated mice, suggesting an increase in myelin thickness, most likely due to remyelination (FIG. 54).

Example 8: Extended therapeutic window data

FIG. 46 and 47 show data relating to the expanded therapeutic window for treatment of a mouse model of CD in males (FIG. 46) and females (FIG. 47). Improved roller performance was observed in treated Nur7 mice compared to untreated Nur7 mice at 52 weeks of age, indicating that ASPA gene therapy is capable of reversing CD pathology.

FIG. 48 shows that working/spatial memory is restored following treatment with the 3rd generation hASPA gene therapy construct in Nur7 mice.

Example 9: Gait analysis data

CatWalk testing was performed using the Noldus CatWalk XT system in a darkened room. Mice were placed inside the CatWalk system and allowed to walk freely to the other end of the CatWalk tunnel. The attached computer records the paw prints and the associated contact time with the illuminated floor, which are then used for the various calculations that generate the data presented. Mice are required to complete at least 5 full paths within the CatWalk and between each path the mice are allowed to turn around for the next path.

Gait analysis data indicate a therapeutic benefit for mice treated with ASPA gene therapy at 6 months and earlier (FIG. 49). Furthermore, gait analysis reveals that mice treated at mature adulthood (p168) continue to benefit from gene therapy treatment (FIG. 50).

Example 10: Additional metabolomic data

This example describes the real-time analysis of increased metabolic activity and oxygen consumption of ASPA-deficient (HEK) cells. Data were generated on a Seahorse XF24 system (Agilent) using approximately 50,000 cells/well. Each sample was run in triplicate or quadruple. For metabolic analysis, the XF Mito Fuel Flex Test (Agilent) or the XF Cell Mito Stress Test (Agilent) were performed.

Mitochondrial stress testing was performed on CRISPR-generated ASPA-deficient HEK WT cells. The data indicate that overall metabolic activity is increased in ASPA-deficient cells (FIG. 51 and 52). An increase in basal respiration was observed, indicating increased oxygen consumption (see Table 3).

Table 3

In addition, ASPA-deficient cells were observed to produce more ATP. For example, FIG. 52 shows mitochondrial stress test data comparing ASPA-deficient and wild-type HEK cells and human oligodendrocytes. The data indicate that wild-type oligodendrocyte cells have a similar percentage energy production profile as wild-type HEK cells. For example, ASPA-deficient HEK cells have a reduced percentage of "spare capacity" or an increased percentage of "proton leak." In contrast, wild-type HEK cells and oligodendrocytes show a much more similar pattern. Interestingly, ASPA-deficient cells exhibit increased non-mitochondrial respiration, indicating that metabolism processed outside of mitochondria is also increased. All data shown are statistically significant with at least n=3 biological replicates.

A Mito flex test was also performed. FIG. 53 shows data indicating that ASPA-deficient cells use more fatty acids and glutamine for energy production. FIG. 53 also shows data pertaining to the dependence, flexibility, and ability of wild-type or ASPA-deficient cells to use glucose (GLC), glutamine (GLN), or fatty acids (FA) for energy production. The data indicate that ASPA-deficient cells are less dependent on glucose for energy production than non-ASPA-deficient cells. In contrast, ASPA-deficient cells are more dependent on fatty acid oxidation to generate energy. Interestingly, the flexibility to use glucose or FA is greater in ASPA-deficient cells. However, the ability to use glucose is reduced in ASPA-deficient cells overall. Lastly, ASPA-deficient cells are dependent on glutamine to generate energy.

Overall, these data are consistent with mouse brain metabolome data (described in Example 1 and FIGS. 55 and 56), where ASPA-deficient brains rely on FA oxidation for energy but do not utilize glucose to the extent of wild-type brains, and that glutamine is a major energy source for the ASPA-deficient brain (e.g., glutamine is reduced in the ASPA-deficient brain).

SEQUENCES

> SEQ ID NO: 1- Codon-optimized human aspartoacylase (hASPA) cDNA (full Kozak sequence underlined)

GCCACCATGACAAGCTGCCACATCGCCGAGGAGCACATCCAGAAAGTCGCCATTTT TGGGGGAACTCACGGTAACGAACTCACAGGGGTCTTCCTGGTGAAGCACTGGCTCG AGAACGGCGCAGAAATCCAGAGAACCGGACTGGAGGTGAAACCCTTCATTACAAA TCCTCGGGCCGTCAAGAAATGCACTCGCTACATCGACTGTGATCTGAACCGGATTTT TGATCTGGAAAATCTCGGCAAGAAAAT GTCCGAGGACCTGCCATACGAAGTGAGGA GAGCTCAGGAGATCAACCACCTCTTCGGACCCAAGGACAGCGAAGATTCCTATGAC ATCATTTTTGATCTGCATAACACCACATCAAATATGGGGTGCACCCTGATCCTCGAG GACAGCCGCAACAATTTCCTGATCCAGATGTTTCACTATATTAAGACAAGTCTGGCA CCACTCCCCTGTTACGTGTATCTGATTGAGCATCCCTCTCTCAAGTACGCTACTACCC GAAGTATCGCAAAATATCCTGTGGGGATTGAAGTCGGTCCTCAGCCACAGGGAGTC CTGCGAGCCGATATCCTCGACCAGATGAGGAAGATGATCAAACATGCTCTGGATTT CATTCACCACTTCAACGAGGGCAAGGAGTTCCCCCTTGCGCCATCGAGGTGTACA AGATCATTGAAAAAGTCGATTATCCTC GGGACGAGAACGGCGAAATTGCCGCTATC ATTCACCCAAATCTGCAGGACCAGGATTGGAAGCCCCTCCATCCTGGGGATCCAAT GTTCCTGACACTCGACGGTAAAACTATCCCACTGGGCGGAGACTGTACCGTGTACC CCGTGTTTGTC AATGAGGCAGCCT ACT ATGAGAAGAAAGAAGCTTTCGCC AAAACA ACAAAACTCACTCTCAATGCTAAATCTATTCGGTGCTGCCTCCACTGA

> SEQ ID NO: 2- Human codon-optimized aspartoacylase (hASPA)MTSCHIAEEHIQKVAIFGGTHGNELTGVFLVKHWLENGAEIQRTGLEVKPFITNPRAVK KCTRYIDCDLNRIFDLENLGKKMSEDLPYEVRRAQEINHLFGPKDSEDSYDIIFDLHNTT SNMGCTLILEDSRNNFLIQMFHYIKTSLAPLPCYVYLIE HPSLKYATTRSIAKYPVGIEVG PQPQGVLRADILDQMRKMIKHALDFIHHFNEGKEFPPCAIEVYKIIEKVDYPRDENGEIA AIIHPNLQDQDWKPLHPGDPMFLTLDGKTIPLGGDCTVYPVFVNEAAYYEKKEAFAKT TKLTLNAKSIRCCLH

>SEQ ID NO:3- Codon-optimized human NAT8L cDNA (full Kozak sequence underlined) GCCACCATGCACTGCGGGCCACCTGATATGGTCTGTGAAACTAAGATTGTCGCTGCC GAGGATCACGAGGCTCTGCCTGGAGCTAAAAAAGATGCTCTGCTGGCCGCCGCCGGCGCCATGTGGCCCCCTCTGCCAGCAGCACCAGGACCAGCAGCAGCACCACCCGCCC CTCCACCCGCCCCTGTGGCCCAGCCACACGGCGGCGCCGGCGGCGCCGGCCCTCCA GGCGGCCGGGGCGTGTGCATCCGGGAGTTCAGAGCAGCAGAGCAGGAGGCAGCAA GGAGAATCTTTTATGACGGCATCATGGAGCGGATCCCCAACACCGCCTTCAGGGGA CTGAGGCAGC ACCCT AGAGCAC AGCTGCTGT ACGC ACTGCTGGCCGCCCTGTGCTTT GCCGTGAGCAGGTCCCTGCTGCTGACCTGTCTGGTGCCCGCCGCCCTGCTGGGACTG AGGTACTATTACAGCCGGAAAGTGATCAGAGCCTATCTGGAGTGCGCCCTGCACAC AGACATGGCCGATATCGAGCAGTATTACATGAAGCCCTGGCTCCTGTTTCTGGGT GGCCGTGCTGGACGGAAACGTGGTGGGAATCGTGGCAGCAAGGGCACACGAGGAG GACAATACCGTGGAGCTGCTGCGCAT GTCTGTGGATAGCAGGTTCCGCGGCAAGGG AATCGCAAAGGCCCTGGGAAGGAAGGTGCTGGAGTTTGCCGTGGTGCACAATTACT CTGCCGTGGTGCTGGGCACCACAGCAGTGAAGGTGGCAGCCCACAAGCTGTATGAG TCCCTGGGCTTTAGGCACATGGGCGCCTCTGATCACTACGTGCTGCCTGGCATGACA CTGTCCCTGGCCGAGAGACTGTTCTTCCAGGTCCGCTACCATAGATATAGACTGCAG CTGAGGGAGGAGTGA

> SEQ ID NO:4- Codon-optimized human NAT8LMHCGPPDMVCETKIVAAEDHEALPGAKKDALLAAAGAMWPPLPAAPGPAAAPPAPPP APVAQPHGGAGGAGPPGGRGVCIREFRAAEQEAARRIFYDGIMERIPNTAFRGLRQHPR AQLLY ALLAALFAVSRSLLLTCLVPAALLGLRYYYSRKVIRA YLEC ALHTDMADIEQY YMKPPGSCFWVAV LDGNVVGIVAARAHEEDNTVELLRMSVDSRFRGKGIAKALGRKV LEFA V VHN Y S A V VLGTT A VKV A AHKLYES LGFRHMG AS DH Y VLPGMTLS LAERLFF Q VRYHRYRLQLREE

Claims (8)

1. A recombinant adeno-associated virus (rAAV) for use in a method of treating Canavan disease, wherein the rAAV comprises: (a) a capsid protein; and (b) a nucleic acid comprising a promoter operably linked to a transgene, wherein the transgene encodes aspartoacylase (ASPA), and wherein the transgene comprises SEQ ID NO:1. 2. The rAAV for use according to claim 1, wherein the rAAV is administered by injection, wherein the administration results in expression of the gene in peripheral tissue, or wherein the administration results in expression of the gene in CNS tissue. 3. The rAAV for use according to claim 2, wherein the injection is selected from the group consisting of intravenous injection, intravascular injection and intraventricular injection. 4. The rAAV for use according to claim 1 or 2, wherein the rAAV is administered intrathecally or intracerebrally. 5. The rAAV for use according to any one of claims 1 to 4, wherein the promoter is an astrocyte-specific promoter, optionally the glial fibrillary acidic protein (GFAP) promoter. 6. The rAAV for use according to any one of claims 1 to 4, wherein the promoter is an improved chicken pactin promoter. 7. The rAAV for use according to any one of claims 1 to 6, wherein the transgene encodes ASPA comprising SEQ ID NO:2. 8. The rAAV for use according to any one of claims 1 to 7, wherein the capsid protein has the AAV9 serotype.